Microorganisms for the production of insect pheromones and related compounds
The present application relates to recombinant microorganisms useful in the biosynthesis of unsaturated C6-C24 fatty alcohols, aldehydes, and acetates which may be useful as insect pheromones, fragrances, flavors, and polymer intermediates. The recombinant microorganisms may express enzymes or enzyme variants useful for production of and/or may be modified to downregulate pathways to shunt production toward unsaturated C6-C24 fatty alcohols, aldehydes, and acetates. The C6-C24 fatty alcohols, aldehydes, and acetates described herein may be used as substrates for metathesis reactions to expand the repertoire of target compounds and pheromones. The application further relates to recombinant microorganisms co-expressing a pheromone pathway and a pathway for the production of a toxic protein, peptide, oligonucleotide, or small molecule suitable for use in an attract-and-kill pest control approach. The application further relates to microorganisms modified to express or downregulate enzymes useful for production of unsaturated short chain fatty alcohols, aldehydes, and acetates which may be useful as insect pheromones, fragrances, flavors, and polymer intermediates. Also provided are methods of producing unsaturated C6-C24 fatty alcohols, aldehydes, and acetates using the recombinant microorganisms, as well as compositions comprising the recombinant microorganisms and/or optionally one or more of the product alcohols, aldehydes, or acetates.
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This application is a continuation of U.S. patent application Ser. No. 16/614,144, filed Nov. 15, 2019 (issued as U.S. Pat. No. 11,104,921 on Aug. 31, 2021), which is a U.S. National Phase of International Patent Application No. PCT/US2018/033151, filed May 17, 2018, which claims priority to U.S. Provisional Application No. 62/507,654, filed May 17, 2017, the contents of each of which are herein incorporated by reference in their entireties.
STATEMENT REGARDING THE SEQUENCE LISTINGThe Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is PRVI_020_02US_SeqList_ST25.txt. The text file is about 240 KB, was created on Aug. 19, 2021, and is being submitted electronically via EFS-Web.
TECHNICAL FIELDThis application relates to recombinant microorganisms useful in the biosynthesis of unsaturated C6-C24 fatty alcohols, aldehydes, and acetates which may be useful as insect pheromones, fragrances, flavors, and polymer intermediates. The application further relates to methods of producing unsaturated C6-C24 fatty alcohols, aldehydes, and acetates using the recombinant microorganisms, as well as compositions comprising one or more of these compounds and/or the recombinant microorganisms.
BACKGROUNDAs the global demand for food grows, there is an increasing need for effective pest control. Conventional insecticides are among the most popular chemical control agents because they are readily available, rapid acting, and highly reliable. However, the overuse, misuse, and abuse of these chemicals have led to resistant pests, alteration of the natural ecology, and in some cases, environmental damage.
The use of insect pheromones to control pest populations has gained increasing popularity as a viable, safe, and environmentally friendly alternative to conventional insecticides. Since their discovery in the late 1950s, these molecules have shown efficacy in reducing insect populations through a variety of methods, including mass trappings, attract and kill, and mating disruption. The latter method in particular represents a non-toxic means of pest control and utilizes the ability of synthetic pheromones to mask naturally occurring pheromones, thereby causing confusion and mating disruption.
Although pheromones have significant potential in agricultural insect control, the cost of synthesizing pheromones using currently available techniques is very high, which prohibits widespread use of this sustainable technology beyond high-value crops. Thus, there is an existing need to develop novel technologies for the cost-efficient production of insect pheromones and related fragrances, flavors, and polymer intermediates. The present inventors address this need with the development of recombinant microorganisms capable of producing a wide-range of unsaturated C6-C24 fatty alcohols, aldehydes, and acetates including synthetic insect pheromones from low-cost feedstocks.
SUMMARY OF THE DISCLOSUREThe present application relates to recombinant microorganisms having a biosynthesis pathway for the production of one or more compounds selected from unsaturated C6-C24 fatty alcohols, aldehydes, and acetates. The recombinant microorganisms described herein may be used for the production of at least one compound, such as an insect pheromone, a fragrance, or a flavoring agent, selected from unsaturated C6-C24 fatty alcohols, aldehydes, and acetates.
In one embodiment, the recombinant microorganism comprises a biosynthesis pathway for the production of an unsaturated C6-C24 fatty aldehyde or fatty alcohol. Accordingly, in a first aspect, the application relates to a recombinant microorganism capable of producing an unsaturated C6-C24 fatty aldehyde or fatty alcohol from an endogenous or exogenous source of saturated C6-C24 fatty acyl-CoA, wherein the recombinant microorganism expresses (a): at least one exogenous nucleic acid molecule encoding a fatty-acyl desaturase that catalyzes the conversion of a saturated C6-C24 fatty acyl-CoA to a corresponding mono- or poly-unsaturated C6-C24 fatty acyl-CoA; and (b): at least one exogenous nucleic acid molecule encoding a fatty aldehyde forming fatty-acyl reductase that catalyzes the conversion of the mono- or poly-unsaturated C6-C24 fatty acyl-CoA from into the corresponding mono- or poly-unsaturated C6-C24 fatty aldehyde. In some embodiments, the mono- or poly-unsaturated C6-C24 fatty aldehyde is an insect pheromone. In some embodiments, the mono- or poly-unsaturated C6-C24 fatty aldehyde is a fragrance or flavoring agent. In some embodiments, the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an acetyl transferase capable of catalyzing the conversion of the mono- or poly-unsaturated C6-C24 fatty alcohol from (b) into a corresponding mono- or poly-unsaturated C6-C24 fatty acetate. (c) at least one exogenous nucleic acid molecule encoding a fatty alcohol forming fatty-acyl reductase that catalyzes the conversion of the mono- or poly-unsaturated C6-C24 fatty acyl-CoA from (a) into the corresponding mono- or poly-unsaturated C6-C24 fatty alcohol. In some embodiments, the mono- or poly-unsaturated C6-C24 fatty alcohol is an insect pheromone. In some embodiments, the mono- or poly-unsaturated C6-C24 fatty alcohol is a fragrance or flavoring agent. In some embodiments, the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an alcohol oxidase or an alcohol dehydrogenase, wherein the alcohol oxidase or alcohol dehydrogenase is capable of catalyzing the conversion of the mono- or poly-unsaturated C6-C24 fatty alcohol from (b) into a corresponding mono- or poly-unsaturated C6-C24 fatty aldehyde. In some embodiments, the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an acetyl transferase capable of catalyzing the conversion of the mono- or poly-unsaturated C6-C24 fatty alcohol from (b) into a corresponding mono- or poly-unsaturated C6-C24 fatty acetate.
In some embodiments, the fatty-acyl desaturase is a desaturase capable of utilizing a fatty acyl-CoA as a substrate that has a chain length of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbon atoms.
In some embodiments, the fatty-acyl desaturase is capable of generating a double bond at position C5, C6, C7, C8, C9, C10, C11, C12, or C13 in the fatty acid or its derivatives, such as, for example, fatty acid CoA esters.
In one exemplary embodiment, the fatty-acyl desaturase is a Z11 desaturase. In various embodiments described herein, the Z11 desaturase, or the nucleic acid sequence that encodes it, can be isolated from organisms of the species Agrotis segetum, Amyelois transitella, Argyrotaenia velutiana, Choristoneura rosaceana, Lampronia capitella, Trichoplusia ni, Helicoverpa zea, or Thalassiosira pseudonana. Further Z11-desaturases, or the nucleic acid sequences encoding them, can be isolated from Bombyx mori, Manduca sexta, Diatraea grandiosella, Earias insulana, Earias vittella, Plutella xylostella, Bombyx mori or Diaphania nitidalis. In exemplary embodiments, the Z11 desaturase comprises a sequence selected from GenBank Accession Nos. JX679209, JX964774, AF416738, AF545481, EU152335, AAD03775, AAF81787, and AY493438. In some embodiments, a nucleic acid sequence encoding a Z11 desaturase from organisms of the species Agrotis segetum, Amyelois transitella, Argyrotaenia velutiana, Choristoneura rosaceana, Lampronia capitella, Trichoplusia ni, Helicoverpa zea, or Thalassiosira pseudonana is codon optimized. In some embodiments, the Z11 desaturase comprises a nucleotide sequence selected from SEQ ID NOs: 9, 18, 24 and 26 from Trichoplusia ni. In some embodiments, the Z11 desaturase comprises an amino acid sequence set forth in SEQ ID NO: 49 from Trichoplusia ni. In other embodiments, the Z11 desaturase comprises a nucleotide sequence selected from SEQ ID NOs: 10 and 16 from Agrotis segetum. In some embodiments, the Z11 desaturase comprises an amino acid sequence set forth in SEQ ID NO: 53 from Agrotis segetum. In some embodiments, the Z11 desaturase comprises a nucleotide sequence selected from SEQ ID NOs: 11 and 23 from Thalassiosira pseudonana. In some embodiments, the Z11 desaturase comprises an amino acid sequence selected from SEQ ID NOs: 50 and 51 from Thalassiosira pseudonana. In certain embodiments, the Z11 desaturase comprises a nucleotide sequence selected from SEQ ID NOs: 12, 17 and 30 from Amyelois transitella. In some embodiments, the Z11 desaturase comprises an amino acid sequence set forth in SEQ ID NO: 52 from Amyelois transitella. In further embodiments, the Z11 desaturase comprises a nucleotide sequence selected from SEQ ID NOs: 13, 19, 25, 27 and 31 from Helicoverpa zea. In some embodiments, the Z11 desaturase comprises an amino acid sequence set forth in SEQ ID NO: 54 from Helicoverpa zea. In some embodiments, the Z11 desaturase comprises an amino acid sequence set forth in SEQ ID NO: 39 from S. inferens. In some embodiments, the Z11 desaturase comprises an amino acid sequence set forth in GenBank Accession nos. AF416738, AGH12217.1, AII21943.1, CAJ43430.2, AF441221, AAF81787.1, AF545481, AJ271414, AY362879, ABX71630.1 and NP001299594.1, Q9N9Z8, ABX71630.1 and AIM40221.1. In some embodiments, the Z11 desaturase comprises a chimeric polypeptide. In some embodiments, a complete or partial Z11 desaturase is fused to another polypeptide. In certain embodiments, the N-terminal native leader sequence of a Z11 desaturase is replaced by an oleosin leader sequence from another species. In certain embodiments, the Z11 desaturase comprises a nucleotide sequence selected from SEQ ID NOs: 15, 28 and 29. In some embodiments, the Z11 desaturase comprises an amino acid sequence selected from SEQ ID NOs: 61, 62, 63, 78, 79 and 80.
In certain embodiments, the Z11 desaturase catalyzes the conversion of a fatty acyl-CoA into a mono- or poly-unsaturated product selected from Z11-13:Acyl-CoA, E11-13:Acyl-CoA, (Z,Z)-7,11-13:Acyl-CoA, Z11-14:Acyl-CoA, E11-14:Acyl-CoA, (E,E)-9,11-14:Acyl-CoA, (E,Z)-9,11-14:Acyl-CoA, (Z,E)-9,11-14:Acyl-CoA, (Z,Z)-9,11-14:Acyl-CoA, (E,Z)-9,11-15:Acyl-CoA, (Z,Z)-9,11-15:Acyl-CoA, Z11-16:Acyl-CoA, E11-16:Acyl-CoA, (E,Z)-6,11-16:Acyl-CoA, (E,Z)-7,11-16:Acyl-CoA, (E,Z)-8,11-16:Acyl-CoA, (E,E)-9,11-16:Acyl-CoA, (E,Z)-9,11-16:Acyl-CoA, (Z,E)-9,11-16:Acyl-CoA, (Z,Z)-9,11-16:Acyl-CoA, (E,E)-11,13-16:Acyl-CoA, (E,Z)-11,13-16:Acyl-CoA, (Z,E)-11,13-16:Acyl-CoA, (Z,Z)-11,13-16:Acyl-CoA, (Z,E)-11,14-16:Acyl-CoA, (E,E,Z)-4,6,11-16:Acyl-CoA, (Z,Z,E)-7,11,13-16:Acyl-CoA, (E,E,Z,Z)-4,6,11,13-16:Acyl-CoA, Z11-17:Acyl-CoA, (Z,Z)-8,11-17:Acyl-CoA, Z11-18:Acyl-CoA, E11-18:Acyl-CoA, (Z,Z)-11,13-18:Acyl-CoA, (E,E)-11,14-18:Acyl-CoA, or combinations thereof.
In another exemplary embodiment, the fatty-acyl desaturase is a Z9 desaturase. In various embodiments described herein, the Z9 desaturase, or the nucleic acid sequence that encodes it, can be isolated from organisms of the species Ostrinia furnacalis, Ostrinia nobilalis, Choristoneura rosaceana, Lampronia capitella, Helicoverpa assulta, or Helicoverpa zea. In exemplary embodiments, the Z9 desaturase comprises a sequence selected from GenBank Accession Nos. AY057862, AF243047, AF518017, EU152332, AF482906, and AAF81788. In some embodiments, a nucleic acid sequence encoding a Z9 desaturase is codon optimized. In some embodiments, the Z9 desaturase comprises a nucleotide sequence set forth in SEQ ID NO: 20 from Ostrinia furnacalis. In some embodiments, the Z9 desaturase comprises an amino acid sequence set forth in SEQ ID NO: 58 from Ostrinia furnacalis. In other embodiments, the Z9 desaturase comprises a nucleotide sequence set forth in SEQ ID NO: 21 from Lampronia capitella. In some embodiments, the Z9 desaturase comprises an amino acid sequence set forth in SEQ ID NO: 59 from Lampronia capitella. In some embodiments, the Z9 desaturase comprises a nucleotide sequence set forth in SEQ ID NO: 22 from Helicoverpa zea. In some embodiments, the Z9 desaturase comprises an amino acid sequence set forth in SEQ ID NO: 60 from Helicoverpa zea. Other Z9 desaturases of the present disclosure include SEQ ID Nos: 95, 97, 99, 101, 103, and 105. In some embodiments, the overexpression of a Z9-18 specific desturase can increase the membrane fluidity to improve the diffusion of fatty alcohols into the supernatant.
In certain embodiments, the Z9 desaturase catalyzes the conversion of a fatty acyl-CoA into a monounsaturated or polyunsaturated product selected from Z9-11:Acyl-CoA, Z9-12:Acyl-CoA, E9-12:Acyl-CoA, (E,E)-7,9-12:Acyl-CoA, (E,Z)-7,9-12:Acyl-CoA, (Z,E)-7,9-12:Acyl-CoA, (Z,Z)-7,9-12:Acyl-CoA, Z9-13:Acyl-CoA, E9-13:Acyl-CoA, (E,Z)-5,9-13:Acyl-CoA, (Z,E)-5,9-13:Acyl-CoA, (Z,Z)-5,9-13:Acyl-CoA, Z9-14:Acyl-CoA, E9-14:Acyl-CoA, (E,Z)-4,9-14:Acyl-CoA, (E,E)-9,11-14:Acyl-CoA, (E,Z)-9,11-14:Acyl-CoA, (Z,E)-9,11-14:Acyl-CoA, (Z,Z)-9,11-14:Acyl-CoA, (E,E)-9,12-14:Acyl-CoA, (Z,E)-9,12-14:Acyl-CoA, (Z,Z)-9,12-14:Acyl-CoA, Z9-15:Acyl-CoA, E9-15:Acyl-CoA, (Z,Z)-6,9-15:Acyl-CoA, Z9-16:Acyl-CoA, E9-16:Acyl-CoA, (E,E)-9,11-16:Acyl-CoA, (E,Z)-9,11-16:Acyl-CoA, (Z,E)-9,11-16:Acyl-CoA, (Z,Z)-9,11-16:Acyl-CoA, Z9-17:Acyl-CoA, E9-18:Acyl-CoA, Z9-18:Acyl-CoA, (E,E)-5,9-18:Acyl-CoA, (E,E)-9,12-18:Acyl-CoA, (Z,Z)-9,12-18:Acyl-CoA, (Z,Z, Z)-3,6,9-18:Acyl-CoA, (E,E,E)-9,12,15-18:Acyl-CoA, (Z,Z, Z)-9,12,15-18:Acyl-CoA, or combinations thereof.
In some embodiments, the recombinant microorganism may express a bifunctional desaturase capable of catalyzing the subsequent desaturation of two double bonds.
In some embodiments, the recombinant microorganism may express more than one exogenous nucleic acid molecule encoding a fatty-acyl desaturase that catalyzes the conversion of a saturated C6-C24 fatty acyl-CoA to a corresponding mono- or poly-unsaturated C6-C24 fatty acyl-CoA. For instance, the recombinant microorganism may express an exogenous nucleic acid molecule encoding a Z11 desaturase and another exogenous nucleic acid molecule encoding a Z9 desaturase.
In some embodiments, the recombinant microorganism may express a fatty-acyl conjugase that acts independently or together with a fatty-acyl desaturase to catalyze the conversion of a saturated or monounsaturated fatty acyl-CoA to a conjugated polyunsaturated fatty acyl-CoA.
In one embodiment, the disclosure provides a recombinant microorganism capable of producing a polyunsaturated C6-C24 aldehyde or fatty alcohol from an endogenous or exogenous source of saturated or monounsaturated C6-C24 fatty acyl-CoA, wherein the recombinant microorganism expresses: (a) at least one exogenous nucleic acid molecule encoding a fatty acyl conjugase that catalyzes the conversion of a saturated or monounsaturated C6-C24 fatty acyl-CoA to a corresponding polyunsaturated C6-C24 fatty acyl-CoA; and (b) at least one exogenous nucleic acid molecule encoding a fatty aldehyde or fatty alcohol forming fatty-acyl reductase that catalyzes the conversion of the polyunsaturated C6-C24 fatty acyl-CoA from (a) into the corresponding polyunsaturated C6-C24 fatty aldehyde or fatty alcohol.
In another embodiment, the recombinant microorganism expresses at least two exogenous nucleic acid molecules encoding fatty-acyl conjugases that catalyze the conversion of a saturated or monounsaturated C6-C24 fatty acyl-CoA to a corresponding polyunsaturated C6-C24 fatty acyl-CoA.
In a further embodiment, the disclosure provides a recombinant microorganism capable of producing a polyunsaturated C6-C24 fatty alcohol from an endogenous or exogenous source of saturated or monounsaturated C6-C24 fatty acyl-CoA, wherein the recombinant microorganism expresses: (a) at least one exogenous nucleic acid molecule encoding a fatty-acyl desaturase and at least one exogenous nucleic acid molecule encoding a fatty acyl conjugase that catalyze the conversion of a saturated or monounsaturated C6-C24 fatty acyl-CoA to a corresponding polyunsaturated C6-C24 fatty acyl-CoA; and (b) at least one exogenous nucleic acid molecule encoding a fatty alcohol forming fatty-acyl reductase that catalyzes the conversion of the polyunsaturated C6-C24 fatty acyl-CoA from (a) into the corresponding polyunsaturated C6-C24 fatty alcohol.
In another embodiment, the recombinant microorganism expresses at least two exogenous nucleic acid molecules encoding fatty-acyl desaturases and at least two exogenous nucleic acid molecules encoding fatty-acyl conjugases that catalyze the conversion of a saturated or monounsaturated C6-C24 fatty acyl-CoA to a corresponding polyunsaturated C6-C24 fatty acyl-CoA.
In yet a further embodiment, the fatty-acyl conjugase is a conjugase capable of utilizing a fatty acyl-CoA as a substrate that has a chain length of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbon atoms.
In certain embodiments, the conjugase, or the nucleic acid sequence that encodes it, can be isolated from organisms of the species Cydia pomonella, Cydia nigricana, Lobesia botrana, Myelois cribrella, Plodia interpunctella, Dendrolimus punctatus, Lampronia capitella, Spodoptera litura, Amyelois transitella, Manduca sexta, Bombyx mori, Calendula officinalis, Trichosanthes kirilowii, Punica granatum, Momordica charantia, Impatiens balsamina, and Epiphyas postvittana. In exemplary embodiments, the conjugase comprises a sequence selected from GenBank Accession No. or Uniprot database: A0A059TBF5, A0A0M3L9E8, A0A0M3L9S4, A0A0M3LAH8, A0A0M3LAS8, A0A0M3LAH8, B6CBS4, XP_013183656.1, XP_004923568.2, ALA65425.1, NP_001296494.1, NP_001274330.1, Q4A181, Q75PL7, Q9FPP8, AY178444, AY178446, AF182521, AF182520, Q95UJ3.
In various embodiments described herein, the fatty alcohol forming acyl-CoA reductase, i.e., fatty alcohol forming fatty-acyl reductase, or the nucleic acid sequence that encodes it, can be isolated from organisms of the species Agrotis segetum, Spodoptera littoralis, Helicoverpa amigera, Spodoptera exigua, Euglena gracilis, or Yponomeuta evonymellus. In exemplary embodiments, the reductase comprises a sequence selected from GenBank Accession Nos. JX679210 and HG423128, and UniProt Accession No. I3PN86. In some embodiments, a nucleic acid sequence encoding a fatty-acyl reductase from organisms of the species Agrotis segetum, Spodoptera littoralis, Helicoverpa amigera, Spodoptera exigua, Euglena gracilis, or Yponomeuta evonymellus is codon optimized. In some embodiments, the reductase comprises a nucleotide sequence set forth in SEQ ID NO: 1 from Agrotis segetum. In some embodiments, the fatty acyl reductase comprises an amino acid sequence set forth in SEQ ID NO: 55 from Agrotis segetum. In other embodiments, the reductase comprises a nucleotide sequence set forth in SEQ ID NO: 2 from Spodoptera littoralis. In some embodiments, the fatty acyl reductase comprises an amino acid sequence set forth in SEQ ID NO: 56 from Spodoptera littoralis. In some embodiments, the reductase comprises a nucleotide sequence selected from SEQ ID NOs: 3, 32, 40, 72, 74, 76 and 81. In some embodiments, the fatty acyl reductase comprises an amino acid sequence set forth in SEQ ID NO: 55 from Agrotis segetum. In other embodiments, the fatty acyl reductase comprises an amino acid sequence set forth in SEQ ID NO: 56 from Spodoptera littoralis. In some embodiments, the fatty acyl reductase comprises an amino acid sequence selected from SEQ ID NOs: 41 and 57 from Helicoverpa armigera. In some embodiments, the fatty acyl reductase comprises an amino acid sequence selected from SEQ ID NOs: 73 and 82 from Spodoptera exigua. In some embodiments, the fatty acyl reductase comprises an amino acid sequence set forth in SEQ ID NO: 75 from Euglena gracilis. In some embodiments, the fatty acyl reductase comprises an amino acid sequence set forth in SEQ ID NO: 77 from Yponomeuta evonymellus.
In some embodiments, the present disclosure teaches using multiple fatty acyl reductase enzymes. In some embodiments, the present disclosure teaches recombinant microorganisms comprising multiple copies of the same fatty acyl reductase. In other embodiments, the present disclosure teaches recombinant microorganisms comprising two or more different fatty acyl reductases. In some embodiments, the different fatty acyl reductases utilize different co-factors. For example, the fatty acyl reductase from Euglena gracilis (SEQ ID NO: 75) uses NADH instead of NADPH as reducing equivalent. In some embodiments, this can allow for co-factor balancing using two or more different reductases.
In some embodiments, the fatty acyl reductase is a mutated fatty acyl reductase and comprises an amino acid sequence selected from SEQ ID NOs: 42-48. In some embodiments, the fatty acyl reductase is a mutated fatty acyl reductase and comprises a nucleotide sequence selected from SEQ ID NOs: 83-89.
In certain embodiments, the fatty alcohol forming fatty-acyl reductase catalyzes the conversion of a mono- or poly-unsaturated fatty acyl-CoA into a fatty alcohol product selected from (Z)-3-hexenol, (Z)-3-nonenol, (Z)-5-decenol, (E)-5-decenol, (Z)-7-dodecenol, (E)-7-dodecenol, (E)-8-dodecenol, (Z)-8-dodecenol, (Z)-9-dodecenol, (E)-9-dodecenol, (Z)-9-tetradecenol, (E)-9-tetradecenol, (Z)-9-hexadecenol, (Z)-11-tetradecenol, (Z)-7-hexadecenol, (Z)-11-hexadecenol, (E)-11-hexadecenol (E)-11-tetradecenol, or (Z,Z)-11,13-hexadecadienol, (11Z,13E)-hexadecadienol, (E,E)-8,10-dodecadienol, (E,Z)-7,9-dodecadienol, (Z)-13-octadecenol, or combinations thereof.
In some embodiments, the recombinant microorganism may express more than one exogenous nucleic acid molecule encoding a fatty alcohol forming fatty-acyl reductase that catalyzes the conversion of a mono- or poly-unsaturated C6-C24 fatty acyl-CoA to a corresponding mono- or poly-unsaturated C6-C24 fatty alcohol.
In a further embodiment, the disclosure provides a recombinant microorganism capable of producing a mono- or poly-unsaturated ≤C18 fatty alcohol from an endogenous or exogenous source of saturated C6-C24 fatty acid, wherein the recombinant microorganism comprises: (a) at least one exogenous nucleic acid molecule encoding a fatty acyl desaturase that catalyzes the conversion of a saturated C6-C24 fatty acyl-CoA to a corresponding mono- or poly-unsaturated C6-C24 fatty acyl-CoA; (b) at least one exogenous nucleic acid molecule encoding an acyl-CoA oxidase that catalyzes the conversion of the mono- or poly-unsaturated C6-C24 fatty acyl-CoA from (a) into a mono- or poly-unsaturated ≤C18 fatty acyl-CoA after one or more successive cycle of acyl-CoA oxidase activity, with a given cycle producing a mono- or poly-unsaturated C4-C22 fatty acyl-CoA intermediate with a two carbon truncation relative to a starting mono- or poly-unsaturated C6-C24 fatty acyl-CoA substrate in that cycle; and (c) at least one exogenous nucleic acid molecule encoding a fatty alcohol forming fatty acyl reductase that catalyzes the conversion of the mono- or poly-unsaturated ≤C18 fatty acyl-CoA from (b) into the corresponding mono- or poly-unsaturated ≤C18 fatty alcohol. In some embodiments, the fatty acyl desaturase is selected from an Argyrotaenia velutinana, Spodoptera litura, Sesamia inferens, Manduca sexta, Ostrinia nubilalis, Helicoverpa zea, Choristoneura rosaceana, Drosophila melanogaster, Spodoptera littoralis, Lampronia capitella, Amyelois transitella, Trichoplusia ni, Agrotis segetum, Ostrinia furnicalis, and Thalassiosira pseudonana derived fatty acyl desaturase. In some embodiments, the fatty acyl desaturase has at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, or 50% sequence identity to a fatty acyl desaturase selected from the group consisting of: SEQ ID NOs: 39, 49-54, 58-63, 78-80 and GenBank Accession nos. AF416738, AGH12217.1, AI121943.1, CAJ43430.2, AF441221, AAF81787.1, AF545481, AJ271414, AY362879, ABX71630.1, NP001299594.1, Q9N9Z8, ABX71630.1 and AIM40221.1. In some embodiments, the acyl-CoA oxidase is selected from Table 5a. In other embodiments, the fatty alcohol forming fatty acyl reductase is selected from an Agrotis segetum, Spodoptera exigua, Spodoptera littoralis, Euglena gracilis, Yponomeuta evonymellus and Helicoverpa armigera derived fatty alcohol forming fatty acyl reductase. In further embodiments, the fatty alcohol forming fatty acyl reductase has at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, or 50% sequence identity to a fatty alcohol forming fatty acyl reductase selected from the group consisting of: SEQ ID NOs: 1-3, 32, 41-48, 55-57, 73, 75, 77 and 82. In some embodiments, the recombinant microorganism is a yeast selected from the group consisting of Yarrowia lipolytica, Saccharomyces cerevisiae, Candida albicans, Candida tropicalis and Candida viswanathii.
In some embodiments, the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an acyltransferase that preferably stores ≤C18 fatty acyl-CoA. In some embodiments, the acyltransferase is selected from the group consisting of glycerol-3-phosphate acyl transferase (GPAT), lysophosphatidic acid acyltransferase (LPAAT), glycerolphospholipid acyltransferase (GPLAT) and diacylglycerol acyltransferases (DGAT). In some preferred embodiments, the acyltransferase is selected from Table 5b.
In some embodiments, the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an acylglycerol lipase that preferably hydrolyzes ester bonds of >C16, of >C14, of >C12 or of >C10 acylglycerol substrates. In some embodiments, the acylglycerol lipase is selected from Table 5c.
In some embodiments, the recombinant microorganism comprises a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous enzymes that catalyzes a reaction in a pathway that competes with the biosynthesis pathway for the production of a mono- or poly-unsaturated ≤C18 fatty alcohol. In further embodiments, the recombinant microorganism comprises a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous enzyme selected from: (i) one or more acyl-CoA oxidase; (ii) one or more acyltransferase; (iii) one or more acylglycerol lipase and/or sterol ester esterase; (iv) one or more (fatty) alcohol dehydrogenase; (v) one or more (fatty) alcohol oxidase; and (vi) one or more cytochrome P450 monooxygenase.
In some preferred embodiments, one or more genes of the microbial host encoding acyl-CoA oxidases are deleted or down-regulated to eliminate or reduce the truncation of desired fatty acyl-CoAs beyond a desired chain-length. In some embodiments, the recombinant microorganism comprises a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous acyl-CoA oxidase enzyme selected from the group consisting of Y. lipolytica POX1 (YALI0E32835g), Y. lipolytica POX2 (YALI0F10857g), Y. lipolytica POX3 (YALI0D24750g), Y. lipolytica POX4 (YALI0E27654g), Y. lipolytica POX5 (YALI0C23859g), Y. lipolytica POX6 (YALI0E06567g); S. cerevisiae POX1 (YGL205W); Candida POX2 (CaO19.1655, CaO19.9224, CTRG_02374, M18259), Candida POX4 (CaO19.1652, CaO19.9221, CTRG_02377, M12160), and Candida POX5 (CaO19.5723, CaO19.13146, CTRG_02721, M12161).
In some embodiments, a recombinant microorganism capable of producing a mono- or poly-unsaturated ≤C18 fatty alcohol, fatty aldehyde and/or fatty acetate from an endogenous or exogenous source of saturated C6-C24 fatty acid is provided, wherein the recombinant microorganism expresses one or more acyl-CoA oxidase enzymes, and wherein the recombinant microorganism is manipulated to delete, disrupt, mutate, and/or reduce the activity of one or more endogenous acyl-CoA oxidase enzymes. In some embodiments, the one or more acyl-CoA oxidase enzymes being expressed are different from the one or more endogenous acyl-CoA oxidase enzymes being deleted or downregulated. In other embodiments, the one or more acyl-CoA oxidase enzymes that are expressed regulate chain length of the mono- or poly-unsaturated ≤C18 fatty alcohol, fatty aldehyde and/or fatty acetate. In other embodiments, the one or more acyl-CoA oxidase enzymes being expressed are selected from Table 5a.
In some embodiments, the recombinant microorganism comprises a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous acyltransferase enzyme selected from the group consisting of Y. lipolytica YALI0C00209g, Y. lipolytica YALI0E18964g, Y. lipolytica YALI0F19514g, Y. lipolytica YALI0C14014g, Y. lipolytica YALI0E16797g, Y. lipolytica YALI0E32769g, and Y. lipolytica YALI0D07986g, S. cerevisiae YBL011w, S. cerevisiae YDL052c, S. cerevisiae YOR175C, S. cerevisiae YPR139C, S. cerevisiae YNR008w, and S. cerevisiae YOR245c, and Candida 1503_02577, Candida CTRG_02630, Candida CaO19.250, Candida CaO19.7881, Candida CTRG_02437, Candida CaO19.1881, Candida CaO19.9437, Candida CTRG_01687, Candida CaO19.1043, Candida CaO19.8645, Candida CTRG_04750, Candida CaO19.13439, Candida CTRG_04390, Candida CaO19.6941, Candida CaO19.14203, and Candida CTRG_06209.
In some embodiments, a recombinant microorganism capable of producing a mono- or poly-unsaturated ≤C18 fatty alcohol, fatty aldehyde and/or fatty acetate from an endogenous or exogenous source of saturated C6-C24 fatty acid is provided, wherein the recombinant microorganism expresses one or more acyltransferase enzymes, and wherein the recombinant microorganism is manipulated to delete, disrupt, mutate, and/or reduce the activity of one or more endogenous acyltransferase enzymes. In some preferred embodiments, one or more genes of the microbial host encoding GPATs, LPAATs, GPLATs and/or DGATs are deleted or downregulated, and replaced with one or more GPATs, LPAATs, GPLATs, or DGATs which prefer to store short-chain fatty acyl-CoAs. In some embodiments, the one or more acyltransferase enzymes being expressed are different from the one or more endogenous acyltransferase enzymes being deleted or downregulated. In other embodiments, the one or more acyltransferase enzymes being expressed are selected from Table 5b.
In some preferred embodiments, one or more genes of the microbial host encoding acylglycerol lipases (mono-, di-, or triacylglycerol lipases) and sterol ester esterases are deleted or downregulated and replaced with one or more acylglycerol lipases which prefer long chain acylglycerol substrates. In some embodiments, the recombinant microorganism comprises a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous acylglycerol lipase and/or sterol ester esterase enzyme selected from the group consisting of Y. lipolytica YALI0E32035g, Y. lipolytica YALI0D17534g, Y. lipolytica YALI0F10010g, Y. lipolytica YALI0C14520g, and Y. lipolytica YALI0E00528g, S. cerevisiae YKL140w, S. cerevisiae YMR313c, S. cerevisiae YKR089c, S. cerevisiae YOR081c, S. cerevisiae YKL094W, S. cerevisiae YLL012W, and S. cerevisiae YLR020C, and Candida CaO19.2050, Candida CaO19.9598, Candida CTRG_01138, Candida W5Q_03398, Candida CTRG_00057, Candida CaO19.5426, Candida CaO19.12881, Candida CTRG_06185, Candida CaO19.4864, Candida CaO19.12328, Candida CTRG_03360, Candida CaO19.6501, Candida CaO19.13854, Candida CTRG_05049, Candida CaO19.1887, Candida CaO19.9443, Candida CTRG_01683, and Candida CTRG_04630.
In some embodiments, the recombinant microorganism comprises a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous cytochrome P450 monooxygenases selected from the group consisting of Y. lipolytica YALI0E25982g (ALK1), Y. lipolytica YALI0F01320g (ALK2), Y. lipolytica YALI0E23474g (ALK3), Y. lipolytica YALI0B13816g (ALK4), Y. lipolytica YALI0B13838g (ALK5), Y. lipolytica YALI0B01848g (ALK6) Y. lipolytica YALI0A15488g (ALK7), Y. lipolytica YALI0C12122g (ALK8), Y. lipolytica YALI0B06248g (ALK9), Y. lipolytica YALI0B20702g (ALK10), Y. lipolytica YALI0C10054g (ALK11) and Y. lipolytica YALI0A20130g (ALK12).
In some embodiments, a recombinant microorganism capable of producing a mono- or poly-unsaturated ≤C18 fatty alcohol, fatty aldehyde and/or fatty acetate from an endogenous or exogenous source of saturated C6-C24 fatty acid is provided, wherein the recombinant microorganism expresses one or more acylglycerol lipase and/or sterol ester esterase enzymes, and wherein the recombinant microorganism is manipulated to delete, disrupt, mutate, and/or reduce the activity of one or more endogenous acylglycerol lipase and/or sterol ester esterase enzymes. In some embodiments, the one or more acylglycerol lipase and/or sterol ester esterase enzymes being expressed are different from the one or more endogenous acylglycerol lipase and/or sterol ester esterase enzymes being deleted or downregulated. In some embodiments, the one or more endogenous or exogenous acylglycerol lipase and/or sterol ester esterase enzymes being expressed prefer to hydrolyze ester bonds of long-chain acylglycerols. In other embodiments, the one or more acylglycerol lipase and/or sterol ester esterase enzymes being expressed are selected from Table 5c.
In some embodiments, the fatty acyl desaturase catalyzes the conversion of a fatty acyl-CoA into a mono- or poly-unsaturated intermediate selected from E5-10:Acyl-CoA, E7-12:Acyl-CoA, E9-14:Acyl-CoA, E11-16:Acyl-CoA, E13-18:Acyl-CoA, Z7-12:Acyl-CoA, Z9-14:Acyl-CoA, Z11-16:Acyl-CoA, Z13-18:Acyl-CoA, Z8-12:Acyl-CoA, Z10-14:Acyl-CoA, Z12-16:Acyl-CoA, Z14-18:Acyl-CoA, Z7-10:Acyl-coA, Z9-12:Acyl-CoA, Z11-14:Acyl-CoA, Z13-16:Acyl-CoA, Z15-18:Acyl-CoA, E7-10:Acyl-CoA, E9-12:Acyl-CoA, E11-14:Acyl-CoA, E13-16:Acyl-CoA, E15-18:Acyl-CoA, E5Z7-12:Acyl-CoA, E7Z9-12:Acyl-CoA, E9Z11-14:Acyl-CoA, E11Z13-16:Acyl-CoA, E13Z15-18:Acyl-CoA, E6E8-10:Acyl-CoA, E8E10-12:Acyl-CoA, E10E12-14:Acyl-CoA, E12E14-16:Acyl-CoA, Z5E8-10:Acyl-CoA, Z7E10-12:Acyl-CoA, Z9E12-14:Acyl-CoA, Z11E14-16:Acyl-CoA, Z13E16-18:Acyl-CoA, Z3-10:Acyl-CoA, Z5-12:Acyl-CoA, Z7-14:Acyl-CoA, Z9-16:Acyl-CoA, Z11-18:Acyl-CoA, Z3Z5-10:Acyl-CoA, Z5Z7-12:Acyl-CoA, Z7Z9-14:Acyl-CoA, Z9Z11-16:Acyl-CoA, Z11Z13-16:Acyl-CoA, and Z13Z15-18:Acyl-CoA. In further embodiments, the mono- or poly-unsaturated ≤C18 fatty alcohol is selected from the group consisting of E5-10:OH, Z8-12:OH, Z9-12:OH, Z11-14:OH, Z11-16:OH, E11-14:OH, E8E10-12:OH, E7Z9-12:OH, Z11Z13-160H, Z9-14:OH, Z9-16:OH, and Z13-18:OH.
In some embodiments, the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an aldehyde forming fatty acyl-CoA reductase capable of catalyzing the conversion of the mono- or poly-unsaturated ≤C18 fatty acid into a corresponding ≤C18 fatty aldehyde. In some preferred embodiments, the aldehyde forming fatty acyl-CoA reductase is selected from the group consisting of Acinetobacter calcoaceticus A0A1C4HN78, A. calcoaceticus N9DA85, A. calcoaceticus R8XW24, A. calcoaceticus A0A1A0GGM5, A. calcoaceticus A0A117N158, and Nostoc punctiforme YP_001865324. In some embodiments, the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an alcohol oxidase or an alcohol dehydrogenase capable of catalyzing the conversion of the mono- or poly-unsaturated ≤C18 fatty alcohol into a corresponding ≤C18 fatty aldehyde. In some preferred embodiments, the ≤C18 fatty aldehyde is selected from the group consisting of Z9-16:Ald, Z11-16:Ald, Z11Z13-16:Ald, and Z13-18:Ald.
In some embodiments, the recombinant microorganism further comprises: at least one endogenous or exogenous nucleic acid molecule encoding an enzyme selected from an alcohol oxidase, an alcohol dehydrogenase capable of catalyzing the conversion of the mono- or poly-unsaturated ≤C18 fatty alcohol into a corresponding ≤C18 fatty aldehyde; and at least one endogenous or exogenous nucleic acid molecule encoding an acetyl transferase capable of catalyzing the conversion of the mono- or poly-unsaturated ≤C18 fatty alcohol into a corresponding ≤C18 fatty acetate. In some preferred embodiments, the mono- or poly-unsaturated ≤C18 fatty aldehyde and ≤C18 fatty acetate is selected from the group consisting of E5-10:Ac, Z7-12:Ac, Z8-12:Ac, Z9-12:Ac, E7Z9-12:Ac, Z9-14:Ac, Z9E12-14:Ac, E11-14:Ac, Z11-14:Ac, Z11-16:Ac, Z9-16:Ac, Z9-16:Ald, Z11-16:Ald, Z11Z13-16:Ald, and Z13-18:Ald.
In some embodiments, the disclosure provides a method of engineering a microorganism that is capable of producing a mono- or poly-unsaturated ≤C18 fatty alcohol from an endogenous or exogenous source of saturated C6-C24 fatty acid, wherein the method comprises introducing into a microorganism the following: (a) at least one exogenous nucleic acid molecule encoding a fatty acyl desaturase that catalyzes the conversion of a saturated C6-C24 fatty acyl-CoA to a corresponding mono- or poly-unsaturated C6-C24 fatty acyl-CoA; (b) at least one exogenous nucleic acid molecule encoding an acyl-CoA oxidase that catalyzes the conversion of the mono- or poly-unsaturated C6-C24 fatty acyl-CoA from (a) into a mono- or poly-unsaturated ≤C18 fatty acyl-CoA after one or more successive cycle of acyl-CoA oxidase activity, with a given cycle producing a mono- or poly-unsaturated C4-C22 fatty acyl-CoA intermediate with a two carbon truncation relative to a starting mono- or poly-unsaturated C6-C24 fatty acyl-CoA substrate in that cycle; and (c) at least one exogenous nucleic acid molecule encoding a fatty alcohol forming fatty acyl reductase that catalyzes the conversion of the mono- or poly-unsaturated ≤C18 fatty acyl-CoA from (b) into the corresponding mono- or poly-unsaturated ≤C18 fatty alcohol. In some embodiments, the microorganism is MATA ura3-302::SUC2 Δpox1 Δpox2 Δpox3 Δpox4 Δpox5 Δpox6 Δfadh Δadh1 Δadh2 Δadh3 Δadh4 Δadh5 Δadh6 Δadh7 Δfao1::URA3.
In some embodiments, the disclosure provides a method of producing a mono- or poly-unsaturated ≤C18 fatty alcohol, fatty aldehyde or fatty acetate from an endogenous or exogenous source of saturated C6-C24 fatty acid, comprising: cultivating a recombinant microorganism described herein in a culture medium containing a feedstock that provides a carbon source adequate for the production of the mono- or poly-unsaturated ≤C18 fatty alcohol, fatty aldehyde or fatty acetate. In some embodiments, the method further comprises a step of recovering the mono- or poly-unsaturated ≤C18 fatty alcohol, fatty aldehyde or fatty acetate. In further embodiments, the recovery step comprises distillation. In yet further embodiments, the recovery step comprises membrane-based separation.
In some embodiments, the mono- or poly-unsaturated ≤C18 fatty alcohol is converted into a corresponding ≤C18 fatty aldehyde using chemical methods. In further embodiments, the chemical methods are selected from TEMPO-bleach, TEMPO-copper-air, TEMPO-PhI(OAc)2, Swern oxidation and noble metal-air. In some embodiments, the mono- or poly-unsaturated ≤C18 fatty alcohol is converted into a corresponding ≤C18 fatty acetate using chemical methods. In further embodiments, the chemical methods utilize a chemical agent selected from the group consisting of acetyl chloride, acetic anhydride, butyryl chloride, butyric anhydride, propanoyl chloride and propionic anhydride in the presence of 4-N,N-dimethylaminopyridine (DMAP) or sodium acetate to esterify the mono- or poly-unsaturated ≤C18 fatty alcohol to the corresponding ≤C18 fatty acetate.
In a further embodiment, the disclosure provides a recombinant Yarrowia lipolytica microorganism capable of producing a mono- or poly-unsaturated C6-C24 fatty alcohol from an endogenous or exogenous source of saturated C6-C24 fatty acid, wherein the recombinant Yarrowia lipolytica microorganism comprises: (a) at least one nucleic acid molecule encoding a fatty acyl desaturase having at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, or 50% sequence identity to a fatty acyl desaturase selected from the group consisting of SEQ ID NOs: 54, 60, 62, 78, 79, 80, 95, 97, 99, 101, 103, and 105 that catalyzes the conversion of a saturated C6-C24 fatty acyl-CoA to a corresponding mono- or poly-unsaturated C6-C24 fatty acyl-CoA; and (b) at least one nucleic acid molecule encoding a fatty alcohol forming fatty acyl reductase having 95% sequence identity to a fatty alcohol forming fatty acyl reductase selected from the group consisting of SEQ ID NOs: 41-48, 57, 73, 75 and 77 that catalyzes the conversion of the mono- or poly-unsaturated C6-C24 fatty acyl-CoA from (a) into the corresponding mono- or poly-unsaturated C6-C24 fatty alcohol.
In some embodiments, the recombinant Yarrowia lipolytica microorganism comprises a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous enzymes that catalyzes a reaction in a pathway that competes with the biosynthesis pathway for the production of a mono- or poly-unsaturated C6-C24 fatty alcohol. In some preferred embodiments, the recombinant Yarrowia lipolytica microorganism comprises a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous enzyme selected from the following: (i) one or more acyl-CoA oxidase selected from the group consisting of YALI0E32835g (POX1), YALI0F10857g (POX2), YALI0D24750g (POX3), YALI0E27654g (POX4), YALI0C23859g (POX5), YALI0E06567g (POX6); (ii) one or more (fatty) alcohol dehydrogenase selected from the group consisting of YALI0F09603g (FADH), YALI0D25630g (ADH1), YALI0E17787g (ADH2), YALI0A16379g (ADH3), YALI0E15818g (ADH4), YALI0D02167g (ADH5), YALI0A15147g (ADH6), YALI0E07766g (ADH7); (iii) a (fatty) alcohol oxidase YALI0B14014g (FAO1); (iv) one or more cytochrome P450 enzyme selected from the group consisting of YALI0E25982g (ALK1), YALI0F01320g (ALK2), YALI0E23474g (ALK3), YALI0B13816g (ALK4), YALI0B13838g (ALK5), YALI0B01848g (ALK6), YALI0A15488g (ALK7), (YALI0C12122g (ALK8), YALI0B06248g (ALK9), YALI0B20702g (ALK10), YALI0C10054g (ALK11) and YALI0A20130g (Alk12); YAS1 (YALI1C03349), Yas2 (YALI0E32417), Gsyl1 (YALI0F18502), HFD1 (YALI0F23793), HFD2 (YALI0E15400), HFD3 (YALI0A17875), HFD4 (YALI0B01298), SDR (YALI0A19536); and (v) one or more diacylglycerol acyltransferase selected from the group consisting of YALI0E32791g (DGA1) and YALI0D07986g (DGA2). In other preferred embodiments, the recombinant Yarrowia lipolytica microorganism comprises a deletion of one or more endogenous enzyme selected from the following: (i) one or more acyl-CoA oxidase selected from the group consisting of YALI0E32835g (POX1), YALI0F10857g (POX2), YALI0D24750g (POX3), YALI0E27654g (POX4), YALI0C23859g (POX5), YALI0E06567g (POX6); (ii) one or more (fatty) alcohol dehydrogenase selected from the group consisting of YALI0F09603g (FADH), YALI0D25630g (ADH1), YALI0E17787g (ADH2), YALI0A16379g (ADH3), YALI0E15818g (ADH4), YALI0D02167g (ADH5), YALI0A15147g (ADH6), YALI0E07766g (ADH7); (iii) a (fatty) alcohol oxidase YALI0B14014g (FAO1); (iv) one or more cytochrome P450 enzyme selected from the group consisting of YALI0E25982g (ALK1), YALI0F01320g (ALK2), YALI0E23474g (ALK3), YALI0B13816g (ALK4), YALI0B13838g (ALK5), YALI0B01848g (ALK6), YALI0A15488g (ALK7), (YALI0C12122g (ALK8), YALI0B06248g (ALK9), YALI0B20702g (ALK10), YALI0C10054g (ALK11) and YALI0A20130g (Alk12); and (v) one or more diacylglycerol acyltransferase selected from the group consisting of YALI0E32791g (DGA1) and YALI0D07986g (DGA2).
In some embodiments, the fatty acyl desaturase catalyzes the conversion of a saturated fatty acyl-CoA into a mono- or poly-unsaturated intermediate selected from Z9-14:Acyl-CoA, Z11-14:Acyl-CoA, E11-14:Acyl-CoA, Z9-16:Acyl-CoA, and Z11-16:Acyl-CoA. In other embodiments, the mono- or poly-unsaturated C6-C24 fatty alcohol is selected from the group consisting of Z9-14:OH, Z11-14:OH, E11-14:OH, Z9-16:OH, Z11-16:OH, Z11Z13-16:OH, and Z13-18:OH.
In some embodiments, the recombinant Yarrowia lipolytica microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an alcohol oxidase or an alcohol dehydrogenase capable of catalyzing the conversion of the mono- or poly-unsaturated C6-C24 fatty alcohol into a corresponding C6-C24 fatty aldehyde. In some embodiments, the alcohol dehydrogenase is selected from Table 3a. In some embodiments, the C6-C24 fatty aldehyde is selected from the group consisting of Z9-14:Ald, Z11-14:Ald, E11-14:Ald, Z9-16:Ald, Z11-16:Ald, Z11Z13-16:Ald and Z13-18:Ald.
In some embodiments, the recombinant Yarrowia lipolytica microorganism further comprises: at least one endogenous or exogenous nucleic acid molecule encoding an alcohol oxidase or an alcohol dehydrogenase capable of catalyzing the conversion of the mono- or poly-unsaturated C6-C24 fatty alcohol into a corresponding C6-C24 fatty aldehyde; and at least one endogenous or exogenous nucleic acid molecule encoding an acetyl transferase capable of catalyzing the conversion of the mono- or poly-unsaturated C6-C24 fatty alcohol into a corresponding C6-C24 fatty acetate. In some embodiments, the mono- or poly-unsaturated C6-C24 fatty aldehyde and C6-C24 fatty acetate is selected from the group consisting of Z9-14:Ac, Z11-14:Ac, E11-14:Ac, Z9-16:Ac, Z11-16:Ac, Z11Z13-16:Ac, Z13-18:Ac, Z9-14:Ald, Z11-14:Ald, E11-14:Ald, Z9-16:Ald, Z11-16:Ald, Z11Z13-16:Ald and Z13-18:Ald.
In some embodiments, the fatty acyl desaturase does not comprise a fatty acyl desaturase comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 64, 65, 66 and 67. In other embodiments, the fatty acyl desaturase does not comprise a fatty acyl desaturase selected from an Amyelois transitella, Spodoptera littoralis, Agrotis segetum, or Trichoplusia ni derived desaturase.
In some embodiments, the disclosure provides a method of engineering a Yarrowia lipolytica microorganism that is capable of producing a mono- or poly-unsaturated C6-C24 fatty alcohol from an endogenous or exogenous source of saturated C6-C24 fatty acid, wherein the method comprises introducing into the Yarrowia lipolytica microorganism the following: (a) at least one nucleic acid molecule encoding a fatty acyl desaturase having at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, or 50% sequence identity to a fatty acyl desaturase selected from the group consisting of SEQ ID NOs: 39, 54, 60, 62, 78, 79, 80, 95, 97, 99, 101, 103, and 105 that catalyzes the conversion of a saturated C6-C24 fatty acyl-CoA to a corresponding mono- or poly-unsaturated C6-C24 fatty acyl-CoA; and (b) at least one nucleic acid molecule encoding a fatty alcohol forming fatty acyl reductase having 95% sequence identity to a fatty alcohol forming fatty acyl reductase selected from the group consisting of SEQ ID NOs: 41-48, 55, 56, 57, 73, 75 and 77 that catalyzes the conversion of the mono- or poly-unsaturated C6-C24 fatty acyl-CoA from (a) into the corresponding mono- or poly-unsaturated C6-C24 fatty alcohol. In some embodiments, the microorganism is MATA ura3-302::SUC2 Apox1 Δpox2 Δpox3 Δpox4 Δpox5 Δpox6 Δfadh Δadh1 Δadh2 Δadh3 Δadh4 Δadh5 Δadh6 Δadh7 Δfao1::URA3.
In some embodiments, the disclosure provides a method of producing a mono- or poly-unsaturated C6-C24 fatty alcohol, fatty aldehyde or fatty acetate from an endogenous or exogenous source of saturated C6-C24 fatty acid, comprising: cultivating a recombinant microorganism described herein in a culture medium containing a feedstock that provides a carbon source adequate for the production of the mono- or poly-unsaturated C6-C24 fatty alcohol, fatty aldehyde or fatty acetate. In some embodiments, the method further comprises a step of recovering the mono- or poly-unsaturated C6-C24 fatty alcohol, fatty aldehyde or fatty acetate. In further embodiments, the recovery step comprises distillation. In yet further embodiments, the recovery step comprises membrane-based separation.
In some embodiments, the mono- or poly-unsaturated C6-C24 fatty alcohol is converted into a corresponding C6-C24 fatty aldehyde using chemical methods. In further embodiments, the chemical methods are selected from TEMPO-bleach, TEMPO-copper-air, TEMPO-PhI(OAc)2, Swern oxidation and noble metal-air. In some embodiments, the mono- or poly-unsaturated C6-C24 fatty alcohol is converted into a corresponding C6-C24 fatty acetate using chemical methods. In further embodiments, the chemical methods utilize a chemical agent selected from the group consisting of acetyl chloride, acetic anhydride, butyryl chloride, butyric anhydride, propanoyl chloride and propionic anhydride in the presence of 4-N,N-dimethylaminopyridine (DMAP) or sodium acetate to esterify the mono- or poly-unsaturated C6-C24 fatty alcohol to the corresponding C6-C24 fatty acetate.
In addition to the biosynthetic pathway described in the first aspect above, the present application provides an additional biosynthetic pathway for the production of an unsaturated C6-C24 fatty alcohol utilizing a saturated C6-C24 fatty acyl-ACP intermediate derived from a C6-C24 fatty acid. Accordingly, in a second aspect, the application relates to a recombinant microorganism capable of producing an unsaturated C6-C24 fatty alcohol from an endogenous or exogenous source of C6-C24 fatty acid, wherein the recombinant microorganism expresses (a): at least one exogenous nucleic acid molecule encoding an acyl-ACP synthetase that catalyzes the conversion of a C6-C24 fatty acid to a corresponding saturated C6-C24 fatty acyl-ACP; (b) at least one exogenous nucleic acid molecule encoding a fatty-acyl-ACP desaturase that catalyzes the conversion of a saturated C6-C24 fatty acyl-ACP to a corresponding mono- or poly-unsaturated C6-C24 fatty acyl-ACP; (c) one or more endogenous or exogenous nucleic acid molecules encoding a fatty acid synthase complex that catalyzes the conversion of the mono- or poly-unsaturated C6-C24 fatty acyl-ACP from (b) to a corresponding mono- or poly-unsaturated C6-C24 fatty acyl-ACP with a two carbon elongation relative to the product of (b); (d): at least one exogenous nucleic acid molecule encoding a fatty aldehyde forming fatty-acyl reductase that catalyzes the conversion of the mono- or poly-unsaturated C6-C24 fatty acyl-ACP from (c) into a corresponding mono- or poly-unsaturated C6-C24 fatty aldehyde; and (e) at least one endogenous or exogenous nucleic acid molecule encoding a dehydrogenase that catalyzes the conversion of the mono- or poly-unsaturated C6-C24 fatty aldehyde C6-C24 from (d) into a corresponding mono- or poly-unsaturated C6-C24 fatty alcohol. In some embodiments, the mono- or poly-unsaturated C6-C24 fatty alcohol is an insect pheromone. In some embodiments, the mono- or poly-unsaturated C6-C24 fatty alcohol is a fragrance or flavoring agent. In some embodiments, the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an alcohol oxidase or an alcohol dehydrogenase, wherein the alcohol oxidase or alcohol dehydrogenase is capable of catalyzing the conversion of the mono- or poly-unsaturated C6-C24 fatty alcohol from (e) into a corresponding mono- or poly-unsaturated C6-C24 fatty aldehyde. In some embodiments, the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an acetyl transferase capable of catalyzing the conversion of the mono- or poly-unsaturated C6-C24 fatty alcohol from (e) into a corresponding mono- or poly-unsaturated C6-C24 fatty acetate.
In some embodiments, acyl-ACP synthetase is a synthetase capable of utilizing a fatty acid as a substrate that has a chain length of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbon atoms.
In various embodiments described herein, the acyl-ACP synthetase, or the nucleic acid that encodes it, can be isolated from organisms of the species Vibrio harveyi, Rhodotorula glutinis, or Yarrowia lipolytica.
In some embodiments, the fatty-acyl-ACP desaturase is a soluble desaturase. In various embodiments described herein, the fatty-acyl-ACP desaturase, or the nucleic acid that encodes it, can be isolated from organisms of the species Pelargonium hortorum, Asclepias syriaca, or Uncaria tomentosa.
In some embodiments, the recombinant microorganism may express more than one exogenous nucleic acid molecule encoding a fatty-acyl desaturase that catalyzes the conversion of a saturated C6-C24 fatty acyl-ACP to a corresponding mono- or poly-unsaturated C6-C24 fatty acyl-ACP.
As described above, fatty acid elongation enzymes, i.e., a fatty acid synthase complex, can be utilized to extend the chain length of a mono- or poly-unsaturated C6-C24 fatty acyl-ACP by two additional carbons at the alpha carbon. In some embodiments, the two additional carbons are derived from endogenous malonyl-CoA. In one embodiment, the one or more nucleic acid molecules encoding a fatty acid synthase complex are endogenous nucleic acid molecules, i.e., the nucleic acid molecule(s) is/are native to the recombinant microorganism. In another embodiment, the one or more nucleic acid molecules encoding a fatty acid synthase complex are exogenous nucleic acid molecules.
In various embodiments described herein, the fatty aldehyde forming acyl-ACP reductase, i.e., fatty aldehyde forming fatty-acyl reductase, or the nucleic acid sequence that encodes it, can be isolated from organisms of the species can be isolated from organisms of the species Pelargonium hortorum, Asclepias syriaca, and Uncaria tomentosa.
As noted above, the recombinant microorganism according to the second aspect comprises at least one endogenous or exogenous nucleic acid molecule encoding a dehydrogenase capable of catalyzing the conversion of the mono- or poly-unsaturated C6-C24 fatty aldehyde from (d) into a corresponding mono- or poly-unsaturated C6-C24 fatty alcohol. In one embodiment, the dehydrogenase is encoded by an endogenous nucleic acid molecule. In another embodiment, the dehydrogenase is encoded by an exogenous nucleic acid molecule. In exemplary embodiments, the endogenous or exogenous nucleic acid molecule encoding a dehydrogenase is isolated from organisms of the species Saccharomyces cerevisiae, Escherichia coli, Yarrowia lipolytica, or Candida tropicalis.
In addition to the biosynthetic pathway described in the first and second aspects above, the present application provides an additional biosynthetic pathway for the production of an unsaturated C6-C24 fatty alcohol utilizing a saturated C6-C24 fatty acyl-ACP intermediate derived from a C6-C24 fatty acid. Accordingly, in a third aspect, the application relates to a recombinant microorganism capable of producing an unsaturated C6-C24 fatty alcohol from an endogenous or exogenous source of C6-C24 fatty acid, wherein the recombinant microorganism expresses (a): at least one exogenous nucleic acid molecule encoding an acyl-ACP synthetase that catalyzes the conversion of a C6-C24 fatty acid to a corresponding saturated C6-C24 fatty acyl-ACP; (b) at least one exogenous nucleic acid molecule encoding a fatty-acyl-ACP desaturase that catalyzes the conversion of a saturated C6-C24 fatty acyl-ACP to a corresponding mono- or poly-unsaturated C6-C24 fatty acyl-ACP; (c) at least one exogenous fatty acyl-ACP thioesterase that catalyzes the conversion of the mono- or poly-unsaturated C6-C24 fatty acyl-ACP from (b) to a corresponding mono- or poly-unsaturated C6-C24 fatty acid; (d) one or more endogenous or exogenous nucleic acid molecules encoding an elongase that catalyzes the conversion of the mono- or poly-unsaturated C6-C24 fatty acyl-CoA derived from CoA activation of the mono- or poly-unsaturated C6-C24 fatty acid from (c) to a corresponding mono- or poly-unsaturated C6-C24 fatty acyl-CoA with a two carbon or greater elongation relative to the product of (c); and (e): at least one exogenous nucleic acid molecule encoding a fatty alcohol forming fatty-acyl reductase that catalyzes the conversion of the mono- or poly-unsaturated C6-C24 fatty acyl-CoA from (d) into a corresponding mono- or poly-unsaturated C6-C24 fatty alcohol. In some embodiments, the mono- or poly-unsaturated C6-C24 fatty alcohol is an insect pheromone. In some embodiments, the mono- or poly-unsaturated C6-C24 fatty alcohol is a fragrance or flavoring agent. In some embodiments, the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an alcohol oxidase or an alcohol dehydrogenase, wherein the alcohol oxidase or alcohol dehydrogenase is capable of catalyzing the conversion of the mono- or poly-unsaturated C6-C24 fatty alcohol from (e) into a corresponding mono- or poly-unsaturated C6-C24 fatty aldehyde. In some embodiments, the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an acetyl transferase capable of catalyzing the conversion of the mono- or poly-unsaturated C6-C24 fatty alcohol from (e) into a corresponding mono- or poly-unsaturated C6-C24 fatty acetate
In some embodiments according to this third aspect, a fatty acyl-ACP thioesterase can be utilized to convert a mono- or poly-unsaturated C6-C24 fatty acyl-ACP into a corresponding mono- or poly-unsaturated C6-C24 fatty acid. In a some embodiments, soluble fatty acyl-ACP thioesterases can be used to release free fatty acids for reactivation to a CoA thioester. Fatty acyl-ACP thioesterases that can be included within the embodiment include, but are not limited to, including Q41635, Q39473, P05521.2, AEM72519, AEM72520, AEM72521, AEM72523, AAC49784, CAB60830, EER87824, EER96252, ABN54268, AAO77182, CAH09236, ACL08376, and homologs thereof may be used. In some embodiments, the mono- or poly-unsaturated C6-C24 fatty acyl-CoA may serve as a substrate for an elongase, which can be utilized to extend the chain length of a mono- or poly-unsaturated C6-C24 fatty acyl-CoA by two additional carbons at the alpha carbon. In some embodiments, the two additional carbons are derived from endogenous malonyl-CoA.
As described above, in some embodiments, the recombinant microorganism according to the first, second, or third aspect further comprises at least one endogenous or exogenous nucleic acid molecule encoding an alcohol oxidase capable of catalyzing the conversion of a mono- or poly-unsaturated C6-C24 fatty alcohol into a corresponding mono- or poly-unsaturated C6-C24 fatty aldehyde. In certain embodiments, the alcohol oxidase, or the nucleic acid sequence that encodes it, can be isolated from organisms of the species Candida boidinii, Komagataella pastoris, Tanacetum vulgare, Simmondsia chinensis, Arabidopsis thaliana, Lotus japonicas, or Candida tropicalis. In exemplary embodiments, the alcohol oxidase comprises a sequence selected from GenBank Accession Nos. Q00922, F2QY27, Q6QIR6, Q8LDPO, and L7VFV2.
As described above, in some embodiments, the recombinant microorganism according to the first or second aspect further comprises at least one endogenous or exogenous nucleic acid molecule encoding an acetyl transferase capable of catalyzing the conversion of a C6-C24 fatty alcohol into a corresponding C6-C24 fatty acetate. In certain embodiments, the acetyl transferase, or the nucleic acid sequence that encodes it, can be isolated from organisms of the species Saccharomyces cerevisiae, Danaus plexippus, Heliotis virescens, Bombyx mori, Agrotis Ipsilon, Agrotis segetum, Euonymus alatus. In exemplary embodiments, the acetyl transferase comprises a sequence selected from GenBank Accession Nos. AY242066, AY242065, AY242064, AY242063, AY242062, EHJ65205, ACX53812, NP_001182381, EHJ65977, EHJ68573, KJ579226, GU594061, KTA99184.1, AIN34693.1, AY605053, XP_002552712.1, XP_503024.1, XP_505595.1, and XP_505513.1.
In alternative embodiments, the fatty alcohol may be converted into a fatty acetate using chemical methods, e.g., via chemical catalysis utilizing a chemical agent such as acetyl chloride, acetic anhydride, butyryl chloride, butyric anhydride, propanoyl chloride and propionic anhydride.
In some embodiments, the recombinant microorganism comprising a biosynthesis pathway for the production of an unsaturated C6-C24 fatty alcohol, aldehyde, or acetate may further be engineered to express one or more nucleic acids encoding protein or polypeptide which, when expressed, is toxic to an insect. Exemplary toxicant producing genes suitable for the present disclosure can be obtained from entomopathogenic organism, such as Bacillus thuringiensis, Pseudomonas aeruginosa, Serratia marcescens, and members of the genus Streptomyces. In an exemplary embodiment, the recombinant microorganism comprising a biosynthesis pathway for the production of an unsaturated C6-C24 fatty alcohol, aldehyde, or acetate may further be engineered to express a nucleic acid encoding a Bacillus thuringiensis (“Bt”) toxin. In additional or alternative embodiments, the recombinant microorganism comprising a biosynthesis pathway for the production of an unsaturated C6-C24 fatty alcohol, aldehyde, or acetate may further be engineered to express a nucleic acid encoding other toxic proteins such as spider venom.
In some embodiments, the recombinant microorganism comprising a biosynthesis pathway for the production of an unsaturated C6-C24 fatty alcohol, aldehyde, or acetate may further be engineered to express an RNAi molecule which, when expressed, produces an oligonucleotide that is toxic to an insect.
In some embodiments, the recombinant microorganism comprising a biosynthesis pathway for the production of an unsaturated C6-C24 fatty alcohol, aldehyde, or acetate may further be engineered to express a metabolic pathway which, when expressed, produces a small molecule that is toxic to an insect. Non-limiting examples of toxic small molecules include azadirachtin, spinosad, avermectin, pyrethrins, and various terpenoids.
In various embodiments described herein, the recombinant microorganism comprising a biosynthesis pathway for the production of an unsaturated C6-C24 fatty alcohol, aldehyde, or acetate may be a eukaryotic microorganism, such as a yeast, a filamentous fungi, or an algae, or alternatively, a prokaryotic microorganism, such as a bacterium. For instance, suitable host cells can include cells of a genus selected from the group consisting of Yarrowia, Candida, Saccharomyces, Pichia, Hansenula, Clostridium, Zymomonas, Escherichia, Salmonella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium, and Streptomyces.
In some embodiments, the recombinant microorganism comprising a biosynthesis pathway for the production of an unsaturated C6-C24 fatty alcohol, aldehyde, or acetate is a yeast. Examples of suitable yeasts include yeasts of a genus selected from the group consisting of Yarrowia, Candida, Saccharomyces, Pichia, Hansenula, Kluyveromyces, Issatchenkia, Zygosaccharomyces, Debaryomyces, Schizosaccharomyces, Pachysolen, Cryptococcus, Trichosporon, Rhodotorula, or Myxozyma. In certain embodiments, the yeast is an oleaginous yeast. Exemplary oleaginous yeasts suitable for use in the present disclosure include members of the genera Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon, and Lipomyces, including, but not limited to the species of Yarrowia lipolytica, Candida tropicalis, Rhodosporidium toruloides, Lipomyces starkey, L. hpoferus, C. revkaufi, C. pulcherrima, C. utilis, Rhodotorula minuta, Trichosporon pullans, T. cutaneum, Cryptococcus curvatus, R. glutinis, and R. graminis.
As will be understood in the art, endogenous enzymes can convert critical substrates and/or intermediates upstream of or within the unsaturated C6-C24 fatty alcohol, aldehyde, or acetate biosynthesis pathway into unwanted by-products. Accordingly, in some embodiments, the recombinant microorganism is manipulated to delete, disrupt, mutate, and/or reduce the activity of one or more endogenous enzymes that catalyzes a reaction in a pathway that competes with the unsaturated C6-C24 fatty alcohol, aldehyde, or acetate biosynthesis pathway.
In one embodiment, the recombinant microorganism is manipulated to delete, disrupt, mutate, and/or reduce the activity of one or more endogenous enzymes that catalyzes the conversion of a fatty acid into a ω-hydroxyfatty acid. In the context of a recombinant yeast microorganism, the recombinant yeast microorganism is engineered to delete, disrupt, mutate, and/or reduce the activity of one or more enzyme selected from XP_504406, XP_504857, XP_504311, XP_500855, XP_500856, XP_500402, XP_500097, XP_501748, XP_500560, XP_501148, XP_501667, XP_500273, BAA02041, CAA39366, CAA39367, BAA02210, BAA02211, BAA02212, BAA02213, BAA02214, AAO73952, AAO73953, AAO73954, AAO73955, AAO73956, AAO73958, AAO73959, AAO73960, AAO73961, AAO73957, XP_002546278, or homologs thereof. In the context of a recombinant bacterial microorganism, the recombinant bacterial microorganism is engineered to delete, disrupt, mutate, and/or reduce the activity of one or more enzyme selected from BAM49649, AAB80867, AAB17462, ADL27534, AAU24352, AAA87602, CAA34612, ABM17701, AAA25760, CAB51047, AAC82967, WP_011027348, or homologs thereof.
In another embodiment, the recombinant microorganism is manipulated to delete, disrupt, mutate, and/or reduce the activity of one or more endogenous enzymes that catalyzes the conversion of a fatty acyl-CoA into α,β-enoyl-CoA. In the context of a recombinant yeast microorganism, the recombinant yeast microorganism is engineered to delete, disrupt, mutate, and/or reduce the activity of one or more enzyme selected from CAA04659, CAA04660, CAA04661, CAA04662, CAA04663, CAG79214, AAA34322, AAA34361, AAA34363, CAA29901, BAA04761, AAA34891, or homologs thereof. In the context of a recombinant bacterial microorganism, the recombinant bacterial microorganism is engineered to delete, disrupt, mutate, and/or reduce the activity of one or more enzyme selected from AAB08643, CAB15271, BAN55749, CAC44516, ADK16968, AEI37634, WP_000973047, WP_025433422, WP_035184107, WP_026484842, CEL80920, WP_026818657, WP_005293707, WP_005883960, or homologs thereof.
In embodiments where the recombinant microorganism is a yeast microorganism, the recombinant microorganism is manipulated to delete, disrupt, mutate, and/or reduce the activity of one or more enzyme involved in peroxisome assembly and/or peroxisome enzyme import. The recombinant yeast microorganism is engineered to delete, disrupt, mutate, and/or reduce the activity of one or more enzyme selected from XP_505754, XP_501986, XP_501311, XP_504845, XP_503326, XP_504029, XP_002549868, XP_002547156, XP_002545227, XP_002547350, XP_002546990, EIW11539, EIW08094, EIW11472, EIW09743, EIW08286, or homologs thereof.
In another embodiment, the recombinant microorganism is manipulated to delete, disrupt, mutate, and/or reduce the activity of one or more endogenous reductase or desaturase enzymes that interferes with the unsaturated C6-C24 fatty alcohol, aldehyde, or acetate, i.e., catalyzes the conversion of a pathway substrate or product into an unwanted by-product.
In another embodiment, the recombinant microorganism is manipulated to delete, disrupt, mutate, and/or reduce the activity of one or more endogenous alcohol oxidase or alcohol dehydrogenase enzymes that catalyzes the unwanted conversion of the desired product, e.g., unsaturated C6-C24 fatty alcohol into a corresponding unsaturated C6-C24 fatty aldehyde.
In another embodiment, the recombinant microorganism is manipulated to delete, disrupt, mutate, and/or reduce the activity of one or more endogenous enzymes that catalyzes a reaction in a pathway that competes with the biosynthesis pathway for one or more unsaturated fatty acyl-CoA intermediates. In one embodiment, the one or more endogenous enzymes comprise one or more diacylglycerol acyltransferases. In the context of a recombinant yeast microorganism, the recombinant yeast microorganism is engineered to delete, disrupt, mutate, and/or reduce the activity of one or more diacylglycerol acyltransferases selected from the group consisting of YALI0E32769g, YALI0D07986g and CTRG_06209, or homolog thereof. In another embodiment, the one or more endogenous enzymes comprise one or more glycerolphospholipid acyltransferases. In the context of a recombinant yeast microorganism, the recombinant yeast microorganism is engineered to delete, disrupt, mutate, and/or reduce the activity of one or more glycerolphospholipid acyltransferases selected from the group consisting of YALI0E16797g and CTG_04390, or homolog thereof. In another embodiment, the one or more endogenous enzymes comprise one or more acyl-CoA/sterol acyltransferases. In the context of a recombinant yeast microorganism, the recombinant yeast microorganism is engineered to delete, disrupt, mutate, and/or reduce the activity of one or more acyl-CoA/sterol acyltransferases selected from the group consisting of YALI0F06578g, CTRG_01764 and CTRG_01765, or homolog thereof.
In another embodiment, the recombinant microorganism is manipulated to delete, disrupt, mutate, and/or reduce the activity of one or more endogenous enzymes that catalyzes a reaction in a pathway that oxidizes fatty aldehyde intermediates. In one embodiment, the one or more endogenous enzymes comprise one or more fatty aldehyde dehydrogenases. In the context of a recombinant yeast microorganism, the recombinant yeast microorganism is engineered to delete, disrupt, mutate, and/or reduce the activity of one or more fatty aldehyde dehydrogenases selected from the group consisting of YALI0A17875g, YALI0E15400g, YALI0B01298g, YALI0F23793g, CTRG_05010 and CTRG_04471, or homolog thereof.
In another embodiment, the recombinant microorganism is manipulated to delete, disrupt, mutate, and/or reduce the activity of one or more endogenous enzymes that catalyzes a reaction in a pathway that consumes fatty acetate products. In one embodiment, the one or more endogenous enzymes comprise one or more sterol esterases. In the context of a recombinant yeast microorganism, the recombinant yeast microorganism is engineered to delete, disrupt, mutate, and/or reduce the activity of one or more sterol esterases selected from the group consisting of YALI0E32035g, YALI0E00528g, CTRG_01138, CTRG_01683 and CTRG_04630, or homolog thereof. In another embodiment, the one or more endogenous enzymes comprise one or more triacylglycerol lipases. In the context of a recombinant yeast microorganism, the recombinant yeast microorganism is engineered to delete, disrupt, mutate, and/or reduce the activity of one or more triacylglycerol lipases selected from the group consisting of YALI0D17534g, YALI0F10010g, CTRG_00057 and CTRG_06185, or homolog thereof. In another embodiment, the one or more endogenous enzymes comprise one or more monoacylglycerol lipases. In the context of a recombinant yeast microorganism, the recombinant yeast microorganism is engineered to delete, disrupt, mutate, and/or reduce the activity of one or more monoacylglycerol lipases selected from the group consisting of YALI0C14520g, CTRG_03360 and CTRG_05049, or homolog thereof. In another embodiment, the one or more endogenous enzymes comprise one or more extracellular lipases. In the context of a recombinant yeast microorganism, the recombinant yeast microorganism is engineered to delete, disrupt, mutate, and/or reduce the activity of one or more extracellular lipases selected from the group consisting of YALI0A20350g, YALI0D19184g, YALI0B09361g, CTRG_05930, CTRG_04188, CTRG_02799, CTRG_03052 and CTRG_03885, or homolog thereof.
In embodiments where the recombinant microorganism is a yeast microorganism, one or more of the exogenous unsaturated C6-C24 fatty alcohol, aldehyde, or acetate pathway genes encodes an enzyme that is localized to a yeast compartment selected from the group consisting of the cytosol, the mitochondria, or the endoplasmic reticulum. In an exemplary embodiment, one or more of the exogenous pathway genes encodes an enzyme that is localized to the endoplasmic reticulum. In another embodiment, at least two exogenous pathway genes encode an enzyme that is localized to the endoplasmic reticulum. In yet another embodiment, all exogenous pathway genes encodes an enzyme that is localized to the endoplasmic reticulum.
In additional embodiments, the present application provides methods of producing an unsaturated C6-C24 fatty alcohol, aldehyde, or acetate using a recombinant microorganism as described herein. In one embodiment, the method includes cultivating the recombinant microorganism in a culture medium containing a feedstock providing a carbon source until the unsaturated C6-C24 fatty alcohol, aldehyde, or acetate is produced and optionally, recovering the unsaturated C6-C24 fatty alcohol, aldehyde, or acetate. Once produced, the unsaturated C6-C24 fatty alcohol, aldehyde, or acetate may be isolated from the fermentation medium using various methods known in the art including, but not limited to, distillation, membrane-based separation gas stripping, solvent extraction, and expanded bed adsorption.
In some embodiments, the recombinant microorganism, e.g., a yeast, may be recovered and produced in dry particulate form. In embodiments involving yeast, the yeast may be dried to produce powdered yeast. In some embodiments, the process for producing powdered yeast comprises spray drying a liquid yeast composition in air, optionally followed by further drying. In some embodiments, the recombinant microorganism composition will comprise the unsaturated C6-C24 fatty alcohol, aldehyde, or acetate when dried.
As described herein, preferred recombinant microorganisms of the disclosure will have the ability to utilize alkanes and fatty acids as carbon sources. However, as will be understood in the art, a variety of carbon sources may be utilized, including but not limited to, various sugars (e.g., glucose, fructose, or sucrose), glycerol, alcohols (e.g., ethanol), organic acids, lignocellulose, proteins, carbon dioxide, carbon monoxide, as well as the aforementioned alkanes and fatty acids. In some embodiments, the recombinant microorganism will convert the carbon source to the unsaturated C6-C24 fatty alcohol, aldehyde, or acetate under aerobic conditions.
As highlighted above, the present application provides methods of producing one or more unsaturated C6-C24 fatty alcohols, aldehydes, or acetates using a recombinant microorganism as described herein. In some embodiments, the product is an insect pheromone. As will be appreciated by the skilled artisan equipped with the instant disclosure, a variety of different exogenous and endogenous enzymes can be expressed in a recombinant host microorganism to produce a desired insect pheromone. Exemplary insect pheromones in the form of fatty alcohols, fatty aldehydes, or fatty acetates capable of being generated using the recombinant microorganisms and methods described herein include, but are not limited to, (Z)-11-hexadecenal, (Z)-11-hexadecenyl acetate, (Z)-9-tetradecenyl acetate, (Z,Z)-11,13-hexadecadienal, (9Z,11E)-hexadecadienal, (E,E)-8,10-dodecadin-1-ol, (7E,9Z)-dodecadienyl acetate, (Z)-3-nonen-1-ol, (Z)-5-decen-1-ol, (Z)-5-decenyl acetate, (E)-5-decen-1-ol, (E)-5-decenyl acetate, (Z)-7-dodecen-1-ol, (Z)-7-dodecenyl acetate, (E)-8-dodecen-1-ol, (E)-8-dodecenyl acetate, (Z)-8-dodecen-1-ol, (Z)-8-dodecenyl acetate, (Z)-9-dodecen-1-ol, (Z)-9-dodecenyl acetate, (Z)-9-tetradecen-1-ol, (Z)-11-tetraceden-1-ol, (Z)-11-tetracedenyl acetate, (E)-11-tetradecen-1-ol, (E)-11-tetradecenyl acetate, (Z)-7-hexadecen-1-ol, (Z)-7-hexadecenal, (Z)-9-hexadecen-1-ol, (Z)-9-hexadecenal, (Z)-9-hexadecenyl acetate, (Z)-11-hexadecen-1-ol, (Z)-13-octadecen-1-ol, (Z)-13-hexadecenyl acetate, and (Z)-13-octadecenayl acetate, and (Z)-13-octadecenal.
In another embodiment of the present application, compositions comprising one of more of the insect pheromone-producing recombinant microorganisms described herein can be provided. In certain embodiments, the composition may further comprise one or more insect pheromones produced by the recombinant microorganism. In further embodiments, the may additionally comprise one or more toxic proteins or polypeptides produced by the recombinant microorganism.
Illustrative embodiments of the disclosure are illustrated in the drawings, in which:
A sequence listing for SEQ ID NO: 1-SEQ ID NO: 105 is part of this application and is incorporated by reference herein. The sequence listing is provided at the end of this document, and is separately provided in computer readable format.
DETAILED DESCRIPTION DefinitionsThe following definitions and abbreviations are to be used for the interpretation of the disclosure.
As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a pheromone” includes a plurality of such pheromones and reference to “the microorganism” includes reference to one or more microorganisms, and so forth.
As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having, “contains,” “containing,” or any other variation thereof, are intended to cover a non-exclusive inclusion. A composition, mixture, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus. Further, unless expressly stated to the contrary, “or” refers to an inclusive “or” and not to an exclusive “or.”
The terms “about” and “around,” as used herein to modify a numerical value, indicate a close range surrounding that explicit value. If “X” were the value, “about X” or “around X” would indicate a value from 0.9X to 1.1X, or, in some embodiments, a value from 0.95X to 1.05X. Any reference to “about X” or “around X” specifically indicates at least the values X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, and 1.05X. Thus, “about X” and “around X” are intended to teach and provide written description support for a claim limitation of, e.g., “0.98X.”
As used herein, the terms “microbial,” “microbial organism,” and “microorganism” include any organism that exists as a microscopic cell that is included within the domains of archaea, bacteria or eukarya, the latter including yeast and filamentous fungi, protozoa, algae, or higher Protista. Therefore, the term is intended to encompass prokaryotic or eukaryotic cells or organisms having a microscopic size and includes bacteria, archaea, and eubacteria of all species as well as eukaryotic microorganisms such as yeast and fungi. Also included are cell cultures of any species that can be cultured for the production of a chemical.
As described herein, in some embodiments, the recombinant microorganisms are prokaryotic microorganism. In some embodiments, the prokaryotic microorganisms are bacteria. “Bacteria”, or “eubacteria”, refers to a domain of prokaryotic organisms. Bacteria include at least eleven distinct groups as follows: (1) Gram-positive (gram+) bacteria, of which there are two major subdivisions: (1) high G+C group (Actinomycetes, Mycobacteria, Micrococcus, others) (2) low G+C group (Bacillus, Clostridia, Lactobacillus, Staphylococci, Streptococci, Mycoplasmas); (2) Proteobacteria, e.g., Purple photosynthetic +non-photosynthetic Gram-negative bacteria (includes most “common” Gram-negative bacteria); (3) Cyanobacteria, e.g., oxygenic phototrophs; (4) Spirochetes and related species; (5) Planctomyces; (6) Bacteroides, Flavobacteria; (7) Chlamydia; (8) Green sulfur bacteria; (9) Green non-sulfur bacteria (also anaerobic phototrophs); (10) Radioresistant micrococci and relatives; (11) Thermotoga and Thermosipho thermophiles.
“Gram-negative bacteria” include cocci, nonenteric rods, and enteric rods. The genera of Gram-negative bacteria include, for example, Neisseria, Spirillum, Pasteurella, Brucella, Yersinia, Francisella, Haemophilus, Bordetella, Escherichia, Salmonella, Shigella, Klebsiella, Proteus, Vibrio, Pseudomonas, Bacteroides, Acetobacter, Aerobacter, Agrobacterium, Azotobacter, Spirilla, Serratia, Vibrio, Rhizobium, Chlamydia, Rickettsia, Treponema, and Fusobacterium.
“Gram positive bacteria” include cocci, nonsporulating rods, and sporulating rods. The genera of gram positive bacteria include, for example, Actinomyces, Bacillus, Clostridium, Corynebacterium, Erysipelothrix, Lactobacillus, Listeria, Mycobacterium, Myxococcus, Nocardia, Staphylococcus, Streptococcus, and Streptomyces.
The term “recombinant microorganism” and “recombinant host cell” are used interchangeably herein and refer to microorganisms that have been genetically modified to express or to overexpress endogenous enzymes, to express heterologous enzymes, such as those included in a vector, in an integration construct, or which have an alteration in expression of an endogenous gene. By “alteration” it is meant that the expression of the gene, or level of a RNA molecule or equivalent RNA molecules encoding one or more polypeptides or polypeptide subunits, or activity of one or more polypeptides or polypeptide subunits is up regulated or down regulated, such that expression, level, or activity is greater than or less than that observed in the absence of the alteration. For example, the term “alter” can mean “inhibit,” but the use of the word “alter” is not limited to this definition. It is understood that the terms “recombinant microorganism” and “recombinant host cell” refer not only to the particular recombinant microorganism but to the progeny or potential progeny of such a microorganism. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
The term “expression” with respect to a gene sequence refers to transcription of the gene and, as appropriate, translation of the resulting mRNA transcript to a protein. Thus, as will be clear from the context, expression of a protein results from transcription and translation of the open reading frame sequence. The level of expression of a desired product in a host cell may be determined on the basis of either the amount of corresponding mRNA that is present in the cell, or the amount of the desired product encoded by the selected sequence. For example, mRNA transcribed from a selected sequence can be quantitated by qRT-PCR or by Northern hybridization (see Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989)). Protein encoded by a selected sequence can be quantitated by various methods, e.g., by ELISA, by assaying for the biological activity of the protein, or by employing assays that are independent of such activity, such as western blotting or radioimmunoassay, using antibodies that recognize and bind the protein. See Sambrook et al., 1989, supra.
The term “polynucleotide” is used herein interchangeably with the term “nucleic acid” and refers to an organic polymer composed of two or more monomers including nucleotides, nucleosides or analogs thereof, including but not limited to single stranded or double stranded, sense or antisense deoxyribonucleic acid (DNA) of any length and, where appropriate, single stranded or double stranded, sense or antisense ribonucleic acid (RNA) of any length, including siRNA. The term “nucleotide” refers to any of several compounds that consist of a ribose or deoxyribose sugar joined to a purine or a pyrimidine base and to a phosphate group, and that are the basic structural units of nucleic acids. The term “nucleoside” refers to a compound (as guanosine or adenosine) that consists of a purine or pyrimidine base combined with deoxyribose or ribose and is found especially in nucleic acids. The term “nucleotide analog” or “nucleoside analog” refers, respectively, to a nucleotide or nucleoside in which one or more individual atoms have been replaced with a different atom or with a different functional group. Accordingly, the term polynucleotide includes nucleic acids of any length, DNA, RNA, analogs and fragments thereof. A polynucleotide of three or more nucleotides is also called nucleotidic oligomer or oligonucleotide.
It is understood that the polynucleotides described herein include “genes” and that the nucleic acid molecules described herein include “vectors” or “plasmids.” Accordingly, the term “gene”, also called a “structural gene” refers to a polynucleotide that codes for a particular sequence of amino acids, which comprise all or part of one or more proteins or enzymes, and may include regulatory (non-transcribed) DNA sequences, such as promoter sequences, which determine for example the conditions under which the gene is expressed. The transcribed region of the gene may include untranslated regions, including introns, 5′-untranslated region (UTR), and 3′-UTR, as well as the coding sequence.
The term “enzyme” as used herein refers to any substance that catalyzes or promotes one or more chemical or biochemical reactions, which usually includes enzymes totally or partially composed of a polypeptide or polypeptides, but can include enzymes composed of a different molecule including polynucleotides.
As used herein, the term “non-naturally occurring,” when used in reference to a microorganism organism or enzyme activity of the disclosure, is intended to mean that the microorganism organism or enzyme has at least one genetic alteration not normally found in a naturally occurring strain of the referenced species, including wild-type strains of the referenced species. Genetic alterations include, for example, modifications introducing expressible nucleic acids encoding metabolic polypeptides, other nucleic acid additions, nucleic acid deletions and/or other functional disruption of the microorganism's genetic material. Such modifications include, for example, coding regions and functional fragments thereof, for heterologous, homologous, or both heterologous and homologous polypeptides for the referenced species. Additional modifications include, for example, non-coding regulatory regions in which the modifications alter expression of a gene or operon. Exemplary non-naturally occurring microorganism or enzyme activity includes the hydroxylation activity described above.
The term “exogenous” as used herein with reference to various molecules, e.g., polynucleotides, polypeptides, enzymes, etc., refers to molecules that are not normally or naturally found in and/or produced by a given yeast, bacterium, organism, microorganism, or cell in nature.
On the other hand, the term “endogenous” or “native” as used herein with reference to various molecules, e.g., polynucleotides, polypeptides, enzymes, etc., refers to molecules that are normally or naturally found in and/or produced by a given yeast, bacterium, organism, microorganism, or cell in nature.
The term “an endogenous or exogenous source of saturated C6-C24 fatty acid” as used herein refers to a source of saturated C6-C24 fatty acid originating from within the microorganism (endogenous), such as when a saturated C6-C24 fatty acid is produced or synthesized inside the microorganism, or originating from outside the microorganism (exogenous), such as when a saturated C6-C24 fatty acid is provided to the microorganism during the course of culturing or cultivating the microorganism in media in flasks or other containers.
The term “heterologous” as used herein in the context of a modified host cell refers to various molecules, e.g., polynucleotides, polypeptides, enzymes, etc., wherein at least one of the following is true: (a) the molecule(s) is/are foreign (“exogenous”) to (i.e., not naturally found in) the host cell; (b) the molecule(s) is/are naturally found in (e.g., is “endogenous to”) a given host microorganism or host cell but is either produced in an unnatural location or in an unnatural amount in the cell; and/or (c) the molecule(s) differ(s) in nucleotide or amino acid sequence from the endogenous nucleotide or amino acid sequence(s) such that the molecule differing in nucleotide or amino acid sequence from the endogenous nucleotide or amino acid as found endogenously is produced in an unnatural (e.g., greater than naturally found) amount in the cell.
As used herein, the term “homologous sequences” “homolog” “homologs” or “orthologs” refers to related sequences (nucleic or amino acid) that are functionally related to the referenced sequence. A functional relationship may be indicated in any one of a number of ways, including, but not limited to: (a) degree of sequence identity and/or (b) the same or similar biological function. Use of the term homolog in this disclosure refers to instances in which both (a) and (b) are indicated. The degree of sequence identity may vary, but in one embodiment, is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, or 50% sequence identity when using standard sequence alignment programs known in the art (e.g., Clustal Omega alignment using default parameters). Homology can be determined using software programs readily available in the art, such as those discussed in Current Protocols in Molecular Biology (F. M. Ausubel et al., eds., 1987) Supplement 30, section 7.718, Table 7.71. Some alignment programs are MacVector (Oxford Molecular Ltd, Oxford, U.K.) and ALIGN Plus (Scientific and Educational Software, Pennsylvania). Other non-limiting alignment programs include Sequencher (Gene Codes, Ann Arbor, Michigan), AlignX, and Vector NTI (Invitrogen, Carlsbad, CA). Thus, a reference to a homolog in the present disclosure will be understood as referencing a related sequence with the same or similar biological function, and a high degree of sequence identity as described above.
The term “fatty acid” as used herein refers to a compound of structure R—COOH, wherein R is a C6 to C24 saturated, unsaturated, linear, branched or cyclic hydrocarbon and the carboxyl group is at position 1. In a particular embodiment, R is a C6 to C24 saturated or unsaturated linear hydrocarbon and the carboxyl group is at position 1.
The term “fatty alcohol” as used herein refers to an aliphatic alcohol having the formula R—OH, wherein R is a C6 to C24 saturated, unsaturated, linear, branched or cyclic hydrocarbon. In a particular embodiment, R is a C6 to C24 saturated or unsaturated linear hydrocarbon.
The term “fatty acyl-CoA” refers to a compound having the structure R—(CO)—S—R1, wherein R1 is Coenzyme A, and the term “fatty acyl-ACP” refers to a compound having the structure R—(CO)—S—R1, wherein R1 is an acyl carrier protein ACP.
The term “short chain” or “short-chain” refers to fatty alcohols, fatty aldehydes, and/or fatty acetates, including pheromones, fragrances, flavors, and polymer intermediates with carbon chain length shorter than or equal to C18.
Introduction
The present disclosure addresses the need for novel technologies for the cost-efficient production of valuable products from low-cost feedstocks. Specifically, the present inventors have addressed this need with the development of recombinant microorganisms capable of producing a wide-range of unsaturated C6-C24 fatty alcohols, aldehydes, and acetates including synthetic insect pheromones, fragrances, flavors, and polymer intermediates from low-cost feedstocks. Thus, aspects of the disclosure are based on the inventors' discovery that recombinant microorganisms can be engineered in order to produce valuable products from low-cost feedstocks, which circumvents conventional synthetic methodologies to produce valuable products.
As discussed above, recombinant microorganisms can be engineered to synthesize mono- or poly-unsaturated C6-C24 fatty alcohols. Mono- or poly-unsaturated C6-C24 fatty alcohols synthesized as described herein can be further converted into the corresponding aldehydes or acetates. Thus, various embodiments of the present disclosure can be used to synthesize a variety of insect pheromones selected from fatty alcohols, aldehydes, and acetates. Additionally, embodiments described herein can also be used for the synthesis of fragrances, flavors, and polymer intermediates.
Engineering of the microbial hosts entail the expression of a non-native pheromone biosynthetic pathway which is comprised of but not limited to one or multiple fatty acyl desaturases, and fatty alcohol-forming or fatty aldehyde-forming reductases. Fatty acids produced by desaturation reactions can be stored intracellularly as triacylglycerides or reduced enzymatically by reductases to form fatty alcohols or aldehydes. Triacylglycerides containing unsaturated fatty acids can be extracted, esterified, and chemically reduced to produce unsaturated fatty alcohols. Fatty alcohols produced via the described pathways can be further converted into fatty aldehyde pheromones, and fatty acetate pheromones via subsequent chemical oxidation, and esterification methods, respectively. Methods of chemical oxidation and esterification are known in the arts. Fatty alcohols produced via the described pheromone biosynthetic pathway can also be further converted into fatty aldehyde pheromones, and fatty acetate pheromones using enzymatic conversion such as alcohol dehydrogenases, and acetyltransferase, respectively. Similarly, fatty acyl-CoA or fatty acyl-ACP formed as intermediates in the pheromone biosynthetic pathway can be released as free fatty acids by native or heterologously derived thioesterases, to become substrates for synthesis of pheromones using metathesis.
Pheromones
As described above, embodiments of the disclosure provide for the synthesis of one or more insect pheromones using a recombinant microorganism. A pheromone is a volatile chemical compound that is secreted by a particular insect for the function of chemical communication within the species. That is, a pheromone is secreted or excreted chemical factor that triggers a social response in members of the same species. There are, inter alia, alarm pheromones, food trail pheromones, sex pheromones, aggregation pheromones, epideictic pheromones, releaser pheromones, primer pheromones, and territorial pheromones, that affect behavior or physiology.
Non-limiting examples of insect pheromones which can be synthesized using the recombinant microorganisms and methods disclosed herein include linear alcohols, aldehydes, and acetates listed in Table 1.
In some aspects, the pheromones synthesized as taught in this disclosure include at least one pheromone listed in Table 2a to modulate the behavior of an insect listed in Table 2a. In other aspects, non-limiting examples of insect pheromones which can be synthesized using the recombinant microorganisms and methods disclosed herein include alcohols, aldehydes, and acetates listed in Table 2a. However, the microorganisms described herein are not limited to the synthesis of C6-C20 pheromones listed in Table 1 and Table 2a. Rather, the disclosed microorganisms can also be utilized in the synthesis of various C6-C24 mono- or poly-unsaturated fatty alcohols, aldehydes, and acetates, including fragrances, flavors, and polymer intermediates.
Most pheromones comprise a hydrocarbon skeleton with the terminal hydrogen substituted by a functional group (Ryan M F (2002). Insect Chemoreception. Fundamental and Applied. Kluwer Academic Publishers). Table 2b shows some common functional groups, along with their formulas, prefixes and suffixes. The presence of one or more double bonds, generated by the loss of hydrogens from adjacent carbons, determines the degree of unsaturation of the molecule and alters the designation of a hydrocarbon from -ane (no multiple bonds) to -ene. The presence of two and three double bonds is indicated by ending the name with -diene and -triene, respectively. The position of each double bond is represented by a numeral corresponding to that of the carbon from which it begins, with each carbon numbered from that attached to the functional group. The carbon to which the functional group is attached is designated -1-. Pheromones may have, but are not limited to, hydrocarbon chain lengths numbering 10 (deca-), 12 (dodeca-), 14 (tetradeca-), 16 (hexadeca-), or 18 (octadeca-) carbons long. The presence of a double bond has another effect. It precludes rotation of the molecule by fixing it in one of two possible configurations, each representing geometric isomers that are different molecules. These are designated either E (from the German word Entgegen, opposite) or Z (Zusammen, together), when the carbon chains are connected on the opposite (trans) or same (cis) side, respectively, of the double bond.
Pheromones described herein can be referred to using IUPAC nomenclature or various abbreviations or variations known to one skilled in the art. For example, (11Z)-hexadecen-1-al, can also be written as Z-11-hexadecen-1-al, Z-11-hexadecenal, or Z-x-y:Ald, wherein x represents the position of the double bond and y represents the number of carbons in the hydrocarbon skeleton. Abbreviations used herein and known to those skilled in the art to identify functional groups on the hydrocarbon skeleton include “Ald,” indicating an aldehyde, “OH,” indicating an alcohol, and “Ac,” indicating an acetyl. Also, the number of carbons in the chain can be indicated using numerals rather than using the written name. Thus, as used herein, an unsaturated carbon chain comprised of sixteen carbons can be written as hexadecene or 16.
Similar abbreviation and derivations are used herein to describe pheromone precursors. For example, the fatty acyl-CoA precursors of (11Z)-hexadecen-1-al can be identified as (11Z)-hexadecenyl-CoA or Z-11-16:Acyl-CoA.
The present disclosure relates to the synthesis of mono- or poly-unsaturated C6-C24 fatty alcohols, aldehydes, and acetates using a recombinant microorganism comprised of one or more heterologous enzymes, which catalyze substrate to product conversions for one or more steps in the synthesis process.
Desaturase
The present disclosure describes enzymes that desaturate fatty acyl substrates to corresponding unsaturated fatty acyl substrates.
In some embodiments, a desaturase is used to catalyze the conversion of a fatty acyl-CoA or acyl-ACP to a corresponding unsaturated fatty acyl-CoA or acyl-ACP. A desaturase is an enzyme that catalyzes the formation of a carbon-carbon double bond in a saturated fatty acid or fatty acid derivative, e.g., fatty acyl-CoA or fatty acyl-ACP (collectively referred to herein as “fatty acyl”), by removing at least two hydrogen atoms to produce a corresponding unsaturated fatty acid/acyl. Desaturases are classified with respect to the ability of the enzyme to selectively catalyze double bond formation at a subterminal carbon relative to the methyl end of the fatty acid/acyl or a subterminal carbon relative to the carbonyl end of the fatty acid/acyl. Omega (ω) desaturases catalyze the formation of a carbon-carbon double bond at a fixed subterminal carbon relative to the methyl end of a fatty acid/acyl. For example, an ω3 desaturase catalyzes the formation of a double bond between the third and fourth carbon relative the methyl end of a fatty acid/acyl. Delta (Δ) desaturases catalyze the formation of a carbon-carbon double bond at a specific position relative to the carboxyl group of a fatty acid or the carbonyl group of a fatty acyl CoA. For example, a Δ9 desaturase catalyzes the formation of a double bond between the C9 and C10 carbons with respect to the carboxyl end of the fatty acid or the carbonyl group of a fatty acyl CoA.
As used herein, a desaturase can be described with reference to the location in which the desaturase catalyzes the formation of a double bond and the resultant geometric configuration (i.e., E/Z) of the unsaturated hydrocarbon. Accordingly, as used herein, a Z9 desaturase refers to a Δ desaturase that catalyzes the formation of a double bond between the C9 and C10 carbons with respect to the carbonyl end of a fatty acid/acyl, thereby orienting two hydrocarbons on opposing sides of the carbon-carbon double bonds in the cis or Z configuration. Similarly, as used herein, a Z11 desaturase refers to a Δ desaturase that catalyzes the formation of a double bond between the C11 and C12 carbons with respect to the carbonyl end of a fatty acid/acyl.
Desaturases have a conserved structural motif. This sequence motif of transmembrane desaturases is characterized by [HX3-4HX7-41(3 non-His)HX2-3(1 nonHis)HHX61-189(40 non-His)HX2-3(1 non-His)HH]. The sequence motif of soluble desaturases is characterized by two occurrences of [D/EEXXH].
In some embodiments, the desaturase is a fatty acyl-CoA desaturase that catalyzes the formation of a double bond in a fatty acyl-CoA. In some such embodiments, the fatty acyl-CoA desaturase described herein is capable of utilizing a fatty acyl-CoA as a substrate that has a chain length of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbon atoms. Thus, the desaturase used in the recombinant microorganism can be selected based on the chain length of the substrate.
In some embodiments, the fatty acyl desaturase described herein is capable of catalyzing the formation of a double bond at a desired carbon relative to the terminal CoA on the unsaturated fatty acyl-CoA. Thus, in some embodiments, a desaturase can be selected for use in the recombinant microorganism which catalyzes double bond insertion at the 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 position with respect to the carbonyl group on a fatty acyl-CoA.
In some embodiments, the fatty acyl desaturase described herein is capable of catalyzing the formation of a double bond in a saturated fatty acyl-CoA such that the resultant unsaturated fatty acyl-CoA has a cis or trans (i.e., Z or E) geometric configuration.
In some embodiments, the desaturase is a fatty acyl-ACP desaturase that catalyzes the formation of a double bond in a fatty acyl-ACP. In some embodiments, the fatty acyl-ACP desaturase described herein is capable of utilizing a fatty acyl-CoA as a substrate that has a chain length of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbon atoms. Thus, the desaturase used in the recombinant microorganism can be selected based on the chain length of the substrate.
In some embodiments, the fatty acyl-ACP desaturase described herein is capable of catalyzing the formation of a double bond at a desired carbon relative to the terminal carbonyl on the unsaturated fatty acyl-ACP. Thus, in some embodiments, a desaturase can be selected for use in the recombinant microorganism which catalyzes double bond insertion at the 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 position with respect to the carbonyl group on a fatty acyl-ACP.
In some embodiments, the fatty acyl desaturase described herein is capable of catalyzing the formation of a double bond in a saturated fatty acyl-CoA such that the resultant unsaturated fatty acyl-ACP has a cis or trans (i.e., Z or E) geometric configuration.
In one embodiment, the fatty acyl desaturase is a Z11 desaturase. In some embodiments, a nucleic acid sequence encoding a Z11 desaturase from organisms of the species Agrotis segetum, Amyelois transitella, Argyrotaenia velutiana, Choristoneura rosaceana, Lampronia capitella, Trichoplusia ni, Helicoverpa zea, or Thalassiosira pseudonana is codon optimized. In some embodiments, the Z11 desaturase comprises a nucleotide sequence selected from SEQ ID NOs: 9, 18, 24 and 26 from Trichoplusia ni. In some embodiments, the Z11 desaturase comprises an amino acid sequence set forth in SEQ ID NO: 49 from Trichoplusia ni. In other embodiments, the Z11 desaturase comprises a nucleotide sequence selected from SEQ ID NOs: 10 and 16 from Agrotis segetum. In some embodiments, the Z11 desaturase comprises an amino acid sequence set forth in SEQ ID NO: 53 from Agrotis segetum. In some embodiments, the Z11 desaturase comprises a nucleotide sequence selected from SEQ ID NOs: 11 and 23 from Thalassiosira pseudonana. In some embodiments, the Z11 desaturase comprises an amino acid sequence selected from SEQ ID NOs: 50 and 51 from Thalassiosira pseudonana. In certain embodiments, the Z11 desaturase comprises a nucleotide sequence selected from SEQ ID NOs: 12, 17 and 30 from Amyelois transitella. In some embodiments, the Z11 desaturase comprises an amino acid sequence set forth in SEQ ID NO: 52 from Amyelois transitella. In further embodiments, the Z11 desaturase comprises a nucleotide sequence selected from SEQ ID NOs: 13, 19, 25, 27 and 31 from Helicoverpa zea. In some embodiments, the Z11 desaturase comprises an amino acid sequence set forth in SEQ ID NO: 54 from Helicoverpa zea. In some embodiments, the Z11 desaturase comprises an amino acid sequence set forth in SEQ ID NO: 39 from S. inferens. In some embodiments, the Z11 desaturase comprises an amino acid sequence set forth in GenBank Accession nos. AF416738, AGH12217.1, AI121943.1, CAJ43430.2, AF441221, AAF81787.1, AF545481, AJ271414, AY362879, ABX71630.1 and NP001299594.1, Q9N9Z8, ABX71630.1 and AIM40221.1. In some embodiments, the Z11 desaturase comprises a chimeric polypeptide. In some embodiments, a complete or partial Z11 desaturase is fused to another polypeptide. In certain embodiments, the N-terminal native leader sequence of a Z11 desaturase is replaced by an oleosin leader sequence from another species. In certain embodiments, the Z11 desaturase comprises a nucleotide sequence selected from SEQ ID NOs: 15, 28 and 29. In some embodiments, the Z11 desaturase comprises an amino acid sequence selected from SEQ ID NOs: 61, 62, 63, 78, 79 and 80.
In one embodiment, the fatty acyl desaturase is a Z9 desaturase. In some embodiments, a nucleic acid sequence encoding a Z9 desaturase is codon optimized. In some embodiments, the Z9 desaturase comprises a nucleotide sequence set forth in SEQ ID NO: 20 from Ostrinia furnacalis. In some embodiments, the Z9 desaturase comprises an amino acid sequence set forth in SEQ ID NO: 58 from Ostrinia furnacalis. In other embodiments, the Z9 desaturase comprises a nucleotide sequence set forth in SEQ ID NO: 21 from Lampronia capitella. In some embodiments, the Z9 desaturase comprises an amino acid sequence set forth in SEQ ID NO: 59 from Lampronia capitella. In some embodiments, the Z9 desaturase comprises a nucleotide sequence set forth in SEQ ID NO: 22 from Helicoverpa zea. In some embodiments, the Z9 desaturase comprises an amino acid sequence set forth in SEQ ID NO: 60 from Helicoverpa zea.
Thus, in some embodiments, the present disclosure teaches a recombinant microorganism comprising a Z11 or Z9 desaturase exhibiting at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, or 50% sequence identity with any one of SEQ ID Nos. selected from the group consisting of 39, 49, 50, 51, 52, 53, 54, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 78, 79, 80, 95, 97, 99, 101, 103, and 105.
Thus, in some embodiments, the present disclosure teaches a recombinant microorganism comprising a nucleic acid molecule encoding for a Z11 or Z9 desaturase, wherein said nucleic acid molecule exhibits at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, or 50% sequence identity with any one of SEQ ID Nos. selected from the group consisting of 9, 10, 11, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 68, 69, 70, 71, 94, 96, 98, 100, 102, and 104.
In some embodiments, the present disclosure teaches a recombinant microorganism comprising at least one nucleic acid molecule encoding a fatty acyl desaturase having at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, or 50% sequence identity to a fatty acyl desaturase selected from the group consisting of SEQ ID NOs: 39, 54, 60, 62, 78, 79, 80, 95, 97, 99, 101, 103, and 105 that catalyzes the conversion of a saturated C6-C24 fatty acyl-CoA to a corresponding mono- or poly-unsaturated C6-C24 fatty acyl-CoA.
Fatty Acyl Reductase
The present disclosure describes enzymes that reduce fatty acyl substrates to corresponding fatty alcohols or aldehydes.
In some embodiments, a fatty alcohol forming fatty acyl-reductase is used to catalyze the conversion of a fatty acyl-CoA to a corresponding fatty alcohol. In some embodiments, a fatty aldehyde forming fatty acyl-reductase is used to catalyze the conversion of a fatty acyl-ACP to a corresponding fatty aldehyde. A fatty acyl reductase is an enzyme that catalyzes the reduction of a fatty acyl-CoA to a corresponding fatty alcohol or the reduction of a fatty acyl-ACP to a corresponding fatty aldehyde. A fatty acyl-CoA and fatty acyl-ACP has a structure of R—(CO)—S—R1, wherein R is a C6 to C24 saturated, unsaturated, linear, branched or cyclic hydrocarbon, and R1 represents CoA or ACP. In a particular embodiment, R is a C6 to C24 saturated or unsaturated linear hydrocarbon. “CoA” is a non-protein acyl carrier group involved in the synthesis and oxidation of fatty acids. “ACP” is an acyl carrier protein, i.e., a polypeptide or protein subunit, of fatty acid synthase used in the synthesis of fatty acids.
Thus, in some embodiments, the disclosure provides for a fatty alcohol forming fatty acyl-reductase which catalyzes the reduction of a fatty acyl-CoA to the corresponding fatty alcohol. For example, R—(CO)—S—CoA is converted to R—CH2OH and CoA-SH when two molecules of NAD(P)H are oxidized to NAD(P)+. Accordingly, in some such embodiments, a recombinant microorganism described herein can include a heterologous fatty alcohol forming fatty acyl-reductase, which catalyzes the reduction a fatty acyl-CoA to the corresponding fatty alcohol. In an exemplary embodiment, a recombinant microorganism disclosed herein includes at least one exogenous nucleic acid molecule encoding a fatty alcohol forming fatty-acyl reductase which catalyzes the conversion of a mono- or poly-unsaturated C6-C24 fatty acyl-CoA into the corresponding mono- or poly-unsaturated C6-C24 fatty alcohol.
In other embodiments, the disclosure provides for a fatty aldehyde forming fatty acyl-reductase which catalyzes the reduction of a fatty acyl-ACP to the corresponding fatty aldehyde. For example, R—(CO)—S-ACP is converted to R—(CO)—H and ACP-SH when one molecule of NAD(P)H is oxidized to NAD(P)+. In some such embodiments, a recombinant microorganism described herein can include a heterologous fatty aldehyde forming fatty acyl-reductase, which catalyzes the reduction a fatty acyl-ACP to the corresponding fatty aldehyde. In an exemplary embodiment, a recombinant microorganism disclosed herein includes at least one exogenous nucleic acid molecule encoding a fatty aldehyde forming fatty-acyl reductase which catalyzes the conversion of a mono- or poly-unsaturated C6-C24 fatty acyl-ACP into the corresponding mono- or poly-unsaturated C6-C24 fatty aldehyde.
In some insect species the respective alcohol-forming fatty acyl reductase (FAR) enzymes are activated via site specific dephosphorylation (Jurenka, R. & Rafaeli, A. Regulatory Role of PBAN in Sex Pheromone Biosynthesis of Heliothine Moths. Front. Endocrinol. (Lausanne). 2: 46 (2011); Gilbert, L. I. Insect Endocrinology. (Academic Press)). Without being bound by any one theory, phosphorylation of heterologously expressed FAR enzymes in yeast such as Y. lipolytica can lead to inactivation, and results in low fatty alcohol titers. In some embodiments, a bioinformatic approach can be used to predict phosphorylated residues within FAR. Alanine substitution of serine and threonine residues has been shown to abolish phosphorylation (Shi, S., Chen, Y., Siewers, V. & Nielsen, J. Improving Production of Malonyl Coenzyme A-Derived Metabolites by Abolishing Snf1-Dependent Regulation of Accl. mBio 5 (2014)). Thus, the impact of alanine substitutions to prevent phosphorylation of serine residues and its impact on fatty alcohol titers can be tested. In addition to alanine substitution, improvement of FAR activity can also be achieved by other amino acid substitutions.
In some embodiments, methods are provided to identify beneficial mutations of FAR based on selection and alteration of phosphorylation-sensitive residues upon its expression in a host microorganism. In a preferred embodiment, the host microorganism is yeast selected from the group consisting of Yarrowia, Candida, Saccharomyces, Pichia, Hansenula, and Kluyveromyces.
Other references for protein phosphorylation sites include: Blom, N., Gammeltoft, S. & Brunak, S. Sequence and structure-based prediction of eukaryotic protein phosphorylation sited. J. Mol. Biol. 294, 1351-1362 (1999); Ingrell, C. R., Miller, M. L., Jensen, O. N. & Blom, N. NetPhosYeast: prediction of protein phosphorylation sites in yeast. Bioinforma. 23: 895-897 (2007); Miller, W. T. Tyrosine kinase signaling and the emergence of multicellularity. Biochim. Biophys. Acta 1823, 1053-1057 (2012), each of which is herein incorporated in its entirety.
In some embodiments, a nucleic acid sequence encoding a fatty-acyl reductase from organisms of the species Agrotis segetum, Spodoptera exigua, Spodoptera littoralis, Euglena gracilis, Yponomeuta evonymellus and Helicoverpa armigera is codon optimized. In some embodiments, the fatty acyl reductase comprises a nucleotide sequence set forth in SEQ ID NO: 1 from Agrotis segetum. In other embodiments, the fatty acyl reductase comprises a nucleotide sequence set forth in SEQ ID NO: 2 from Spodoptera littoralis. In some embodiments, the fatty acyl reductase comprises a nucleotide sequence selected from SEQ ID NOs: 3, 32, 40, 72, 74, 76 and 81. In some embodiments, the fatty acyl reductase comprises an amino acid sequence set forth in SEQ ID NO: 55 from Agrotis segetum. In other embodiments, the fatty acyl reductase comprises an amino acid sequence set forth in SEQ ID NO: 56 from Spodoptera littoralis. In some embodiments, the fatty acyl reductase comprises an amino acid sequence selected from SEQ ID NOs: 41 and 57 from Helicoverpa armigera. In some embodiments, the fatty acyl reductase comprises an amino acid sequence selected from SEQ ID NOs: 73 and 82 from Spodoptera exigua. In some embodiments, the fatty acyl reductase comprises an amino acid sequence set forth in SEQ ID NO: 75 from Euglena gracilis. In some embodiments, the fatty acyl reductase comprises an amino acid sequence set forth in SEQ ID NO: 77 from Yponomeuta evonymellus.
In some embodiments, the production of unsaturated fatty alcohols in a recombinant microorganism comprises the expression of one or more mutant FARs. In certain embodiments, Helicoverpa amigera fatty acyl-CoA reductase (HaFAR) variants are provided which have increased enzymatic activity relative to enzymatic activity of a wild type Helicoverpa amigera fatty acyl-CoA reductase encoded by an amino acid sequence set forth in SEQ ID NO: 41. In some embodiments, the increased enzymatic activity is a net activity increase in amount of fatty alcohol produced relative to the amount of fatty alcohol produced by a wild type enzymatic activity of HaFAR encoded by an amino acid sequence set forth in SEQ ID NO: 41. In some embodiments, a wild type HaFAR comprises a nucleotide sequence set forth in SEQ ID NO: 90. In some embodiments, a variant of a wild type HaFAR encoded by an amino acid sequence set forth in SEQ ID NO: 41 comprises point mutations at the following positions: 560X, 5195X, 5298X, 5378X, 5394X, 5418X, and 5453X, wherein X comprises an amino acid selected from F, L, M, I, V, P, T, A, Y, K, H, N, Q, K, D, E, C, W and R. In some embodiments, a variant of a wild type HaFAR encoded by an amino acid sequence set forth in SEQ ID NO: 41 comprises a combination of point mutations selected from mutations at the following amino acid positions: 560X, S195X, S298X, S378X, S394X, 5418X, and 5453X, wherein X comprises an amino acid selected from F, L, M, I, V, P, T, A, Y, K, H, N, Q, K, D, E, C, W and R. In some embodiments, the fatty acyl reductase is a mutated fatty acyl reductase and comprises an amino acid sequence selected from SEQ ID NOs: 42-48. In some embodiments, the fatty acyl reductase is a mutated fatty acyl reductase and comprises a nucleotide sequence selected from SEQ ID NOs: 83-89.
Thus, in some embodiments, the present disclosure teaches a recombinant microorganism comprising a fatty acyl reductase exhibiting at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, or 50% sequence identity with any one of SEQ ID Nos. selected from the group consisting of, 41, 42, 43, 44, 45, 46, 47, 48, 55, 56, 57, 73, 75, 77, and 82.
Thus, in some embodiments, the present disclosure teaches a recombinant microorganism comprising a nucleic acid molecule encoding for a fatty acyl reductase, wherein said nucleic acid molecule exhibits at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, or 50% sequence identity with any one of SEQ ID Nos. selected from the group consisting of 1, 2, 3, 32, 37, 40, 72, 74, 76, 81, 83, 84, 85, 86, 87, 88, 89, and 90.
In some embodiments, the present disclosure teaches a recombinant microorganism comprising at least one nucleic acid molecule encoding a fatty acyl reductase having at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, or 50% sequence identity to a fatty acyl reductase selected from the group consisting of SEQ ID NOs: 41-48, 57, 73, 75 and 77 that catalyzes the conversion of the mono- or poly-unsaturated C6-C24 fatty acyl-CoA into the corresponding mono- or poly-unsaturated C6-C24 fatty alcohol.
Acyl-ACP Synthetase
The present disclosure describes enzymes that ligate a fatty acid to the corresponding fatty acyl-ACP.
In some embodiments, an acyl-ACP synthetase is used to catalyze the conversion of a fatty acid to a corresponding fatty acyl-ACP. An acyl-ACP synthetase is an enzyme capable of ligating a fatty acid to ACP to produce a fatty acid acyl-ACP. In some embodiments, an acyl-ACP synthetase can be used to catalyze the conversion of a fatty acid to a corresponding fatty acyl-ACP. In some embodiments, the acyl-ACP synthetase is a synthetase capable of utilizing a fatty acid as a substrate that has a chain length of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbon atoms. In one such embodiment, a recombinant microorganism described herein can include a heterologous acyl-ACP synthetase, which catalyzes the conversion of a fatty acid to a corresponding fatty acyl-ACP. In an exemplary embodiment, a recombinant microorganism disclosed herein includes at least one exogenous nucleic acid molecule which encodes an acyl-ACP synthetase that catalyzes the conversion of a saturated C6-C24 fatty acid to a corresponding saturated C6-C24 fatty acyl-ACP.
Fatty Acid Synthase Complex
The present disclosure describes enzymes that catalyze the elongation of a carbon chain in fatty acid.
In some embodiments, a fatty acid synthase complex is used to catalyze initiation and elongation of a carbon chain in a fatty acid. A “fatty acid synthase complex” refers to a group of enzymes that catalyzes the initiation and elongation of a carbon chain on a fatty acid. The ACP along with the enzymes in the fatty acid synthase (FAS) pathway control the length, degree of saturation, and branching of the fatty acids produced. The steps in this pathway are catalyzed by enzymes of the fatty acid biosynthesis (fab) and acetyl-CoA carboxylase (acc) gene families. Depending upon the desired product, one or more of these genes can be attenuated, expressed or over-expressed. In exemplary embodiments, one or more of these genes is over-expressed.
There are two principal classes of fatty acid synthases. Type I (FAS I) systems utilize a single large, multifunctional polypeptide and are common to both mammals and fungi (although the structural arrangement of fungal and mammalian synthases differ). The Type I FAS system is also found in the CMN group of bacteria (corynebacteria, mycobacteria, and nocardia). The Type II FAS (FAS II) is characterized by the use of discrete, monofunctional enzymes for fatty acid synthesis, and is found in archaea and bacteria.
The mechanism of FAS I and FAS II elongation and reduction is the substantially similar, as the domains of the FAS I multienzyme polypeptides and FAS II enzymes are largely conserved.
Fatty acids are synthesized by a series of decarboxylative Claisen condensation reactions from acetyl-CoA and malonyl-CoA. The steps in this pathway are catalyzed by enzymes of the fatty acid biosynthesis (fab) and acetyl-CoA carboxylase (acc) gene families. For a description of this pathway, see, e.g., Heath et al., Prog. Lipid Res. 40:467, 2001, which is herein incorporated by reference in its entirety. Without being limited by theory, in bacteria, acetyl-CoA is carboxylated by acetyl-CoA carboxylase (Acc, a multi-subunit enzyme encoded by four separate genes, accABCD), to form malonyl-CoA. In yeast, acetyl-CoA is carboxylated by the yeast equivalents of the acetyl-CoA carboxylase, encoded by ACC1 and ACC2. In bacteria, the malonate group is transferred to ACP by malonyl-CoA:ACP transacylase (FabD) to form malonyl-ACP. In yeast, a malonyl-palmityl tranferase domain adds malonyl from malonyl-CoA to the ACP domain of the FAS complex. A condensation reaction then occurs, where malonyl-ACP merges with acyl-CoA, resulting in β-ketoacyl-ACP. In this manner, the hydrocarbon substrate is elongated by 2 carbons.
Following elongation, the β-keto group is reduced to the fully saturated carbon chain by the sequential action of a keto-reductase (KR), dehydratase (DH), and enol reductase (ER). The elongated fatty acid chain is carried between these active sites while attached covalently to the phosphopantetheine prosthetic group of ACP. First, the β-ketoacyl-ACP is reduced by NADPH to form β-hydroxyacyl-ACP. In bacteria, this step is catalyzed by β-ketoacyl-ACP reductase (FabG). The equivalent yeast reaction is catalyzed by the ketoreductase (KR) domain of FAS. β-hydroxyacyl-ACP is then dehydrated to form trans-2-enoyl-ACP, which is catalyzed by either β-hydroxyacyl-ACP dehydratase/isomerase (FabA) or β-hydroxyacyl-ACP dehydratase (FabZ) in bacteria or the dehydratase (DH) domain of FAS in yeast. NADPH-dependent trans-2-enoyl-ACP reductase I, II, or III (Fabl, FabK, and FabL, respectively) in bacteria and the enol reductase (ER) domain of FAS in yeast reduces trans-2-enoyl-ACP to form acyl-ACP. Subsequent cycles are started by the condensation of malonyl-ACP with acyl-ACP by β-ketoacyl-ACP synthase I or β-ketoacyl-ACP synthase II (FabB and FabF, respectively, in bacteria or the beta-ketoacyl synthase (KS) domain in yeast).
In some embodiments, a fatty acid synthase complex can be used to catalyze elongation of a fatty acyl-ACP to a corresponding fatty acyl-ACP with a two carbon elongation relative to the substrate.
Dehydrogenase
The present disclosure describes enzymes that catalyze the conversion of a fatty aldehyde to a fatty alcohol. In some embodiments, an alcohol dehydrogenase (ADH, Table 3 and Table 3a) is used to catalyze the conversion of a fatty aldehyde to a fatty alcohol. A number of ADHs identified from alkanotrophic organisms, Pseudomonas fluorescens NRRL B-1244 (Hou et al. 1983), Pseudomonas butanovora ATCC 43655 (Vangnai and Arp 2001), and Acinetobacter sp. strain M-1 (Tani et al. 2000), have shown to be active on short to medium-chain alkyl alcohols (C2 to C14). Additionally, commercially available ADHs from Sigma, Horse liver ADH and Baker's yeast ADH have detectable activity for substrates with length Cm and greater. The reported activities for the longer fatty alcohols may be impacted by the difficulties in solubilizing the substrates. For the yeast ADH from Sigma, little to no activity is observed for C12 to C14 aldehydes by (Tani et al. 2000), however, activity for C12 and C16 hydroxy-w-fatty acids has been observed (Lu et al. 2010). Recently, two ADHs were characterized from Geobacillus thermodenitrificans NG80-2, an organism that degrades C15 to C36 alkanes using the LadA hydroxylase. Activity was detected from methanol to 1-triacontanol (C30) for both ADHs, with 1-octanol being the preferred substrate for ADH2 and ethanol for ADH1 (Liu et al. 2009).
The use of ADHs in whole-cell bioconversions has been mostly focused on the production of chiral alcohols from ketones (Ernst et al. 2005) (Schroer et al. 2007). Using the ADH from Lactobacillus brevis and coupled cofactor regeneration with isopropanol, Schroer et al. reported the production of 797 g of (R)-methyl-3 hydroxybutanoate from methyl acetoacetate, with a space time yield of 29 g/L/h (Schroer et al. 2007). Examples of aliphatic alcohol oxidation in whole-cell transformations have been reported with commercially obtained S. cerevisiae for the conversion of hexanol to hexanal (Presecki et al. 2012) and 2-heptanol to 2-heptanone (Cappaert and Larroche 2004).
In some embodiments, the present disclosure teaches a recombinant microorganism comprising a deletion, disruption, mutation, and or reduction in the activity of one or more endogenous (fatty) alcohol dehydrogenase selected from the group consisting of YALI0F09603g (FADH), YALI0D25630g (ADH1), YALI0E17787g (ADH2), YALI0A16379g (ADH3), YALI0E15818g (ADH4), YALI0D02167g (ADH5), YALI0A15147g (ADH6), YALI0E07766g (ADH7).
Thus, in some embodiments, the recombinant microorganism of the present disclosure will comprise deletions or other disruptions in endogenous genes encoding proteins exhibiting at least 100%, 99%, 98%, 97%, 95%, 94%, 93%, 92%, 91%, or 90% sequence identity with the proteins encoded by YALI0F09603g (FADH), YALI0D25630g (ADH1), YALI0E17787g (ADH2), YALI0A16379g (ADH3), YALI0E15818g (ADH4), YALI0D02167g (ADH5), YALI0A15147g (ADH6), and YALI0E07766g (ADH7).
Thus, in some embodiments, the recombinant microorganism of the present disclosure will comprise deletions in endogenous genes encoding proteins exhibiting at least 100%, 99%, 98%, 97%, 95%, 94%, 93%, 92%, 91%, or 90% sequence identity with uniprot database IDs Q6C297 (FADH), Q6C7T0 (ADH1), F2Z678 (ADH2), Q6CGT5 (ADH3), Q6C5R5 (ADH4), Q6CAT5 (ADH5), Q6CGX5 (ADH6), and Q6C7K3 (ADH7).
Alcohol Oxidase
The present disclosure describes enzymes that oxidize fatty alcohols to fatty aldehydes.
In some embodiments, an alcohol oxidase (AOX) is used to catalyze the conversion of a fatty alcohol to a fatty aldehyde. Alcohol oxidases catalyze the conversion of alcohols into corresponding aldehydes (or ketones) with electron transfer via the use of molecular oxygen to form hydrogen peroxide as a by-product. AOX enzymes utilize flavin adenine dinucleotide (FAD) as an essential cofactor and regenerate with the help of oxygen in the reaction medium. Catalase enzymes may be coupled with the AOX to avoid accumulation of the hydrogen peroxide via catalytic conversion into water and oxygen.
Based on the substrate specificities, AOXs may be categorized into four groups: (a) short chain alcohol oxidase, (b) long chain alcohol oxidase, (c) aromatic alcohol oxidase, and (d) secondary alcohol oxidase (Goswami et al. 2013). Depending on the chain length of the desired substrate, some members of these four groups are better suited than others as candidates for evaluation.
Short chain alcohol oxidases (including but not limited to those currently classified as EC 1.1.3.13, Table 4) catalyze the oxidation of lower chain length alcohol substrates in the range of C1-C8 carbons (van der Klei et al. 1991) (Ozimek et al. 2005). Aliphatic alcohol oxidases from methylotrophic yeasts such as Candida boidinii and Komagataella pastoris (formerly Pichia pastoris) catalyze the oxidation of primary alkanols to the corresponding aldehydes with a preference for unbranched short-chain aliphatic alcohols. The most broad substrate specificity is found for alcohol oxidase from the Pichia pastoris including propargyl alcohol, 2-chloroethanol, 2-cyanoethanol (Dienys et al. 2003). The major challenge encountered in alcohol oxidation is the high reactivity of the aldehyde product. Utilization of a two liquid phase system (water/solvent) can provide in-situ removal of the aldehyde product from the reaction phase before it is further converted to the acid. For example, hexanal production from hexanol using Pichia pastoris alcohol oxidase coupled with bovine liver catalase was achieved in a bi-phasic system by taking advantage of the presence of a stable alcohol oxidase in aqueous phase (Karra-Chaabouni et al. 2003). For example, alcohol oxidase from Pichia pastoris was able to oxidize aliphatic alcohols of C6 to C11 when used biphasic organic reaction system (Murray and Duff 1990). Methods for using alcohol oxidases in a biphasic system according to (Karra-Chaabouni et al. 2003) and (Murray and Duff 1990) are incorporated by reference in their entirety.
Long chain alcohol oxidases (including but not limited to those currently classified as EC 1.1.3.20; Table 5) include fatty alcohol oxidases, long chain fatty acid oxidases, and long chain fatty alcohol oxidases that oxidize alcohol substrates with carbon chain length of greater than six (Goswami et al. 2013). Banthorpe et al. reported a long chain alcohol oxidase purified from the leaves of Tanacetum vulgare that was able to oxidize saturated and unsaturated long chain alcohol substrates including hex-trans-2-en-1-ol and octan-1-ol (Banthorpe 1976) (Cardemil 1978). Other plant species, including Simmondsia chinensis (Moreau, R. A., Huang 1979), Arabidopsis thaliana (Cheng et al. 2004), and Lotus japonicas (Zhao et al. 2008) have also been reported as sources of long chain alcohol oxidases. Fatty alcohol oxidases are mostly reported from yeast species (Hommel and Ratledge 1990) (Vanhanen et al. 2000) (Hommel et al. 1994) (Kemp et al. 1990) and these enzymes play an important role in long chain fatty acid metabolism (Cheng et al. 2005). Fatty alcohol oxidases from yeast species that degrade and grow on long chain alkanes and fatty acid catalyze the oxidation of fatty alcohols. Fatty alcohol oxidase from Candida tropicalis has been isolated as microsomal cell fractions and characterized for a range of substrates (Eirich et al. 2004) (Kemp et al. 1988) (Kemp et al. 1991) (Mauersberger et al. 1992). Significant activity is observed for primary alcohols of length C8 to C16 with reported KM in the 10-50 μM range (Eirich et al. 2004). Alcohol oxidases described may be used for the conversion of medium chain aliphatic alcohols to aldehydes as described, for example, for whole-cells Candida boidinii (Gabelman and Luzio 1997), and Pichia pastoris (Duff and Murray 1988) (Murray and Duff 1990). Long chain alcohol oxidases from filamentous fungi were produced during growth on hydrocarbon substrates (Kumar and Goswami 2006) (Savitha and Ratledge 1991). The long chain fatty alcohol oxidase (LjFAO1) from Lotus japonicas has been heterologously expressed in E. coli and exhibited broad substrate specificity for alcohol oxidation including 1-dodecanol and 1-hexadecanol (Zhao et al. 2008).
In some embodiments, the present disclosure teaches a recombinant microorganism comprising a deletion, disruption, mutation, and or reduction in the activity of one or more endogenous a (fatty) alcohol oxidase YALI0B14014g (FAO1).
Thus, in some embodiments, the recombinant microorganism of the present disclosure will comprise deletions or other disruptions in endogenous genes encoding proteins exhibiting at least 100%, 99%, 98%, 97%, 95%, 94%, 93%, 92%, 91%, or 90% sequence identity with the proteins encoded by a (fatty) alcohol oxidase YALI0B14014g (FAO1)
Thus, in some embodiments, the recombinant microorganism of the present disclosure will comprise deletions in endogenous genes encoding proteins exhibiting at least 100%, 99%, 98%, 97%, 95%, 94%, 93%, 92%, 91%, or 90% sequence identity with uniprot database IDs Q6CEP8 (FAO1).
Acetyl Transferase
The present disclosure describes enzymes that convert alcohols to fatty acetates.
In some embodiments, an acetyl transferase is used to catalyze the conversion of a fatty alcohol to a fatty acetate. An acetyl transferase is an enzyme that has the ability to produce an acetate ester by transferring the acetyl group from acetyl-CoA to an alcohol. In some embodiments, the acetyl transferase may have an EC number of 2.3.1.84.
The acetyl transferase, or the nucleic acid sequence that encodes it, can be isolated from various organisms, including but not limited to, organisms of the species Candida glabrata, Saccharomyces cerevisiae, Danaus plexippus, Heliotis virescens, Bombyx mori, Agrotis Ipsilon, Agrotis segetum, Euonymus alatus, Homo sapiens, Lachancea thermotolerans and Yarrowia lipolytica. In exemplary embodiments, the acetyl transferase comprises a sequence selected from GenBank Accession Nos. AY242066, AY242065, AY242064, AY242063, AY242062, EHJ65205, ACX53812, NP_001182381, EHJ65977, EHJ68573, KJ579226, GU594061, KTA99184.1, AIN34693.1, AY605053, XP_002552712.1, XP_503024.1, and XP_505595.1, and XP_505513.1. Exemplary acetyl transferase enzymes are listed in Table 5d. Additional exemplary acetyl transferase peptides may be found in US2010/0199548, which is herein incorporated by reference.
Fatty Acyl-ACP Thioesterase
Acyl-ACP thioesterase releases free fatty acids from Acyl-ACPs, synthesized from de novo fatty acid biosynthesis. The reaction terminates fatty acid biosynthesis. In plants, fatty acid biosynthesis occurs in the plastid and thus requires plastid-localized acyl-ACP thioesterases. The main products of acyl-ACP thioesterase are oleate (C18:0) and to a lesser extent palmitate (C16:0) in the vegetative tissues of all plants. The released free fatty acids are re-esterified to coenzyme A in the plastid envelope and exported out of plastid.
There are two isoforms of acyl-ACP thioesterase, FatA and FatB. Substrate specificity of these isoforms determines the chain length and level of saturated fatty acids in plants. The highest activity of FatA is with C18:1-ACP. FatA has very low activities towards other acyl-ACPs when compared with C18:1-ACP. FatB has highest activity with C16:0-ACP. It also has significant high activity with C18:1-ACP, followed by C18:0-ACP and C16:1-ACP. Kinetics studies of FatA and FatB indicate that their substrate specificities with different acyl-ACPs came from the Kcat values, rather than from Km. Km values of the two isoforms with different substrates are similar, in the micromolar order. Domain swapping of FatA and FatB indicates the N-terminus of the isoforms determines their substrate specificities (Salas J J and Ohlrogge J B (2002) Characterization of substrate specificity of plant FatA and FatB acyl-ACP thioesterases. Arch Biochem Biophys 403(1): 25-34). For those plants which predominantly accumulate medium-chain length saturated fatty acids in seeds, they evolved with specialized FatB and/or FatA thioesterases (Voelker T and Kinney A J (2001) Variations in the biosynthesis of seed-storage lipids. Annu Rev Plant Physiol Plant Mol Biol 52: 335-361). For example, laurate (12:0) is the predominant seed oil in coconut. Correspondingly, the medium-chain specific acyl-ACP thioesterase activity was detected in coconut seeds.
In some embodiments, the present disclosure teaches a recombinant microorganism comprising a deletion, disruption, mutation, and or reduction in the activity of YALI0E16016g (FAT1).
Thus, in some embodiments, the recombinant microorganism of the present disclosure will comprise deletions or other disruptions in endogenous genes encoding proteins exhibiting at least 100%, 99%, 98%, 97%, 95%, 94%, 93%, 92%, 91%, or 90% sequence identity with the protein encoded by YALI0E16016g (FAT1).
Thus, in some embodiments, the recombinant microorganism of the present disclosure will comprise deletions in endogenous genes encoding proteins exhibiting at least 100%, 99%, 98%, 97%, 95%, 94%, 93%, 92%, 91%, or 90% sequence identity with uniprot database ID Q6C5Q8 (FAO1).
Acyl-CoA Oxidase
Acyl-CoA oxidase (ACO) acts on CoA derivatives of fatty acids with chain lengths from 8 to 18. They are flavoenzymes containing one noncovalently bound FAD per subunit and belong to the same superfamily as mitochondrial acyl-CoA dehydrogenases. Like mitochondrial fatty acyl-CoA dehydrogenases, peroxisomal acyl-CoA oxidases catalyze the initial and rate-determining step of the peroxisomal fatty acid β-oxidation pathway, i.e. α,β-dehydrogenation of acyl-CoA, yielding trans-2-enoyl-CoA in the reductive half-reaction. In the oxidative half-reaction of peroxisomal acyl-CoA oxidase, the reduced FAD is reoxidized by molecular oxygen, producing hydrogen peroxide.
Acyl-CoA oxidase is a homodimer and the polypeptide chain of the subunit is folded into the N-terminal alpha-domain, beta-domain, and C-terminal alpha-domain. Functional differences between the peroxisomal acyl-CoA oxidases and the mitochondrial acyl-CoA dehydrogenases are attributed to structural differences in the FAD environments.
In some embodiments, recombinant microorganisms and methods are provided for the production of short chain fatty alcohols, fatty aldehydes and/or fatty acetates. In certain embodiments, the short chain fatty alcohols, fatty aldehydes and/or fatty acetates have carbon chain length shorter than or equal to C16. In some embodiments, the short chain fatty alcohols, fatty aldehydes and/or fatty acetates are produced from long chain fatty acids. In some preferred embodiments of methods to produce short chain pheromones, select enzymes capable of shortening fatty acyl-CoAs in the pheromone biosynthetic pathway are co-expressed with pheromone biosynthetic pathway enzymes. Examples of suitable chain shortening enzymes include FAD-dependent acyl-CoA oxidase. In the case of fatty acid molecules with an even number of carbons, chain shortening enzymes produce a molecule of acetyl-CoA, and a fatty acyl-CoA shortened by two carbons. Fatty acid molecules with an odd number of carbons are oxidized in a similar fashion producing acetyl-CoA molecules during every round of oxidation until the chain-length is reduced to 5 carbons. In the final cycle of oxidation, this 5-carbon acyl-CoA is oxidized to produce acetyl-CoA and propionyl-CoA.
It is known that acyl-CoA oxidases exhibit varying specificity towards substrates with different chain-length (
In a further embodiment, the disclosure provides a recombinant microorganism capable of producing a mono- or poly-unsaturated ≤C18 fatty alcohol from an endogenous or exogenous source of saturated C6-C24 fatty acid, wherein the recombinant microorganism comprises: (a) at least one exogenous nucleic acid molecule encoding a fatty acyl desaturase that catalyzes the conversion of a saturated C6-C24 fatty acyl-CoA to a corresponding mono- or poly-unsaturated C6-C24 fatty acyl-CoA; (b) at least one exogenous nucleic acid molecule encoding an acyl-CoA oxidase that catalyzes the conversion of the mono- or poly-unsaturated C6-C24 fatty acyl-CoA from (a) into a mono- or poly-unsaturated ≤C18 fatty acyl-CoA after one or more successive cycle of acyl-CoA oxidase activity, with a given cycle producing a mono- or poly-unsaturated C4-C22 fatty acyl-CoA intermediate with a two carbon truncation relative to a starting mono- or poly-unsaturated C6-C24 fatty acyl-CoA substrate in that cycle; and (c) at least one exogenous nucleic acid molecule encoding a fatty alcohol forming fatty acyl reductase that catalyzes the conversion of the mono- or poly-unsaturated ≤C18 fatty acyl-CoA from (b) into the corresponding mono- or poly-unsaturated ≤C18 fatty alcohol. In some embodiments, the fatty acyl desaturase is selected from an Argyrotaenia velutinana, Spodoptera litura, Sesamia inferens, Manduca sexta, Ostrinia nubilalis, Helicoverpa zea, Choristoneura rosaceana, Drosophila melanogaster, Spodoptera littoralis, Lampronia capitella, Amyelois transitella, Trichoplusia ni, Agrotis segetum, Ostrinia furnicalis, and Thalassiosira pseudonana derived fatty acyl desaturase. In some embodiments, the fatty acyl desaturase has at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, or 50% sequence identity to a fatty acyl desaturase selected from the group consisting of: SEQ ID NOs: 39, 49-54, 58-63, 78-80 and GenBank Accession nos. AF416738, AGH12217.1, AI121943.1, CAJ43430.2, AF441221, AAF81787.1, AF545481, AJ271414, AY362879, ABX71630.1, NP001299594.1, Q9N9Z8, ABX71630.1 and AIM40221.1. In some embodiments, the acyl-CoA oxidase is selected from Table 5a. In other embodiments, the fatty alcohol forming fatty acyl reductase is selected from an Agrotis segetum, Spodoptera exigua, Spodoptera littoralis, Euglena gracilis, Yponomeuta evonymellus and Helicoverpa armigera derived fatty alcohol forming fatty acyl reductase. In further embodiments, the fatty alcohol forming fatty acyl reductase has at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, or 50% sequence identity to a fatty alcohol forming fatty acyl reductase selected from the group consisting of: SEQ ID NOs: 1-3, 32, 41-48, 55-57, 73, 75, 77 and 82. In some embodiments, the recombinant microorganism is a yeast selected from the group consisting of Yarrowia lipolytica, Saccharomyces cerevisiae, Candida albicans, Candida tropicalis and Candida viswanathii.
In some preferred embodiments of methods to produce fatty alcohols, fatty aldehydes and/or fatty acetates, one or more genes of the microbial host encoding acyl-CoA oxidases are deleted or down-regulated to eliminate or reduce the truncation of desired fatty acyl-CoAs beyond a desired chain-length. Such deletion or down-regulation targets include but are not limited to Y. lipolytica POX1(YALI0E32835g), Y. lipolytica POX2(YALI0F10857g), Y. lipolytica POX3(YALI0D24750g), Y. lipolytica POX4(YALI0E27654g), Y. lipolytica POX5(YALI0C23859g), Y. lipolytica POX6(YALI0E06567g); S. cerevisiae POX1(YGL205W); Candida POX2 (CaO19.1655, CaO19.9224, CTRG_02374, M18259), Candida POX4 (CaO19.1652, CaO19.9221, CTRG_02377, M12160), and Candida POX5 (CaO19.5723, CaO19.13146, CTRG_02721, M12161).
In some embodiments, the present disclosure teaches a recombinant microorganism comprising a deletion, disruption, mutation, and or reduction in the activity of one or more acyl-CoA oxidases selected from the group consisting of POX1(YALI0E32835g), Y. lipolytica POX2(YALI0F10857g), Y. lipolytica POX3(YALI0D24750g), Y. lipolytica POX4(YALI0E27654g), Y. lipolytica POX5(YALI0C23859g), Y. lipolytica POX6(YALI0E06567g).
Thus, in some embodiments, the recombinant microorganism of the present disclosure will comprise deletions or other disruptions in endogenous genes encoding proteins exhibiting at least 100%, 99%, 98%, 97%, 95%, 94%, 93%, 92%, 91%, or 90% sequence identity with the proteins encoded by POX1 (YALI0E32835g), Y. lipolytica POX2 (YALI0F10857g), Y. lipolytica POX3 (YALI0D24750g), Y. lipolytica POX4 (YALI0E27654g), Y. lipolytica POX5 (YALI0C23859g), Y. lipolytica POX6 (YALI0E06567g).
Thus, in some embodiments, the recombinant microorganism of the present disclosure will comprise deletions in endogenous genes encoding proteins exhibiting at least 100%, 99%, 98%, 97%, 95%, 94%, 93%, 92%, 91%, or 90% sequence identity with uniprot database ID POX1(074934), Y. lipolytica POX2 (074935), Y. lipolytica POX3 (074936), Y. lipolytica POX4 (F2Z627), Y. lipolytica POX5 (F2Z630), Y. lipolytica POX6 (Q6C6T0).
In some embodiments, a recombinant microorganism capable of producing a mono- or poly-unsaturated ≤C18 fatty alcohol, fatty aldehyde and/or fatty acetate from an endogenous or exogenous source of saturated C6-C24 fatty acid is provided, wherein the recombinant microorganism expresses one or more acyl-CoA oxidase enzymes, and wherein the recombinant microorganism is manipulated to delete, disrupt, mutate, and/or reduce the activity of one or more endogenous acyl-CoA oxidase enzymes. In some embodiments, the one or more acyl-CoA oxidase enzymes being expressed are different from the one or more endogenous acyl-CoA oxidase enzymes being deleted or downregulated. In some embodiments, the recombinant microorganism further expresses pheromone biosynthetic pathway enzymes. In further embodiments, the pheromone biosynthetic pathway enzymes comprise one or more fatty acyl desaturase and/or fatty acyl conjugase. In yet further embodiments, the pheromone biosynthetic pathway enzymes comprise one or more fatty alcohol forming fatty acyl reductase. In some embodiments, the one or more acyl-CoA oxidase enzymes that are expressed are selected from Table 5a. In other embodiments, the one or more acyl-CoA oxidase enzymes that are expressed regulate chain length of the mono- or poly-unsaturated ≤C18 fatty alcohol, fatty aldehyde and/or fatty acetate. In some embodiments, the one or more endogenous acyl-CoA oxidase enzymes that are deleted, disrupted, mutated, or downregulated are selected from Y. lipolytica POX1(YALI0E32835g), Y. lipolytica POX2(YALI0F10857g), Y. lipolytica POX3(YALI0D24750g), Y. lipolytica POX4(YALI0E27654g), Y. lipolytica POX5(YALI0C23859g), Y. lipolytica POX6(YALI0E06567g); S. cerevisiae POX1(YGL205W); Candida POX2 (CaO19.1655, CaO19.9224, CTRG_02374, M18259), Candida POX4 (CaO19.1652, CaO19.9221, CTRG_02377, M12160), and Candida POX5 (CaO19.5723, CaO19.13146, CTRG_02721, M12161). In other embodiments, the one or more endogenous acyl-CoA oxidase enzymes that are deleted, disrupted, mutated, or downregulated control chain length of the mono- or poly-unsaturated ≤C18 fatty alcohol, fatty aldehyde and/or fatty acetate.
In some embodiments, a method of producing a mono- or poly-unsaturated ≤C18 fatty alcohol, fatty aldehyde and/or fatty acetate from an endogenous or exogenous source of saturated C6-C24 fatty acid are provided, wherein the method comprises introducing into or expressing in a recombinant microorganism at least one endogenous or exogenous nucleic acid molecule encoding an acyl-CoA oxidase and introducing a deletion, insertion, or loss of function mutation in one or more gene encoding an acyl-CoA oxidase, wherein the at least one endogenous or exogenous nucleic acid molecule encoding an acyl-CoA oxidase being introduced or expressed is different from the one or more gene encoding an acyl-CoA oxidase being deleted or downregulated. In some embodiments, the method further comprises introducing into or expressing in the recombinant microorganism at least one endogenous or exogenous nucleic acid molecule encoding a fatty acyl desaturase and/or fatty acyl conjugase. In further embodiments, the method further comprises introducing into or expressing in the recombinant microorganism at least one endogenous or exogenous nucleic acid molecule encoding a fatty alcohol forming fatty acyl reductase. In some embodiments, the least one endogenous or exogenous nucleic acid molecule encodes an acyl-CoA oxidase selected from Table 5a. In other embodiments, the least one endogenous or exogenous nucleic acid molecule encodes an acyl-CoA oxidase that regulates chain length of the mono- or poly-unsaturated C6-C24 fatty alcohol, fatty aldehyde and/or fatty acetate. In some embodiments, the one or more gene being deleted or downregulated encodes an acyl-CoA oxidase selected from Y. lipolytica POX1(YALI0E32835g), Y. lipolytica POX2(YALI0F10857g), Y. lipolytica POX3(YALI0D24750g), Y. lipolytica POX4(YALI0E27654g), Y. lipolytica POX5(YALI0C23859g), Y. lipolytica POX6(YALI0E06567g); S. cerevisiae POX1(YGL205W); Candida POX2 (CaO19.1655, CaO19.9224, CTRG_02374, M18259), Candida POX4 (CaO19.1652, CaO19.9221, CTRG_02377, M12160), and Candida POX5 (CaO19.5723, CaO19.13146, CTRG_02721, M12161). In other embodiments, the one or more gene being deleted or downregulated encodes an acyl-CoA oxidase that regulates chain length of the mono- or poly-unsaturated C6-C24 fatty alcohol, fatty aldehyde and/or fatty acetate.
Acyl Transferases
In some embodiments, recombinant microorganisms and methods are provided for the production of short chain fatty alcohols, fatty aldehydes and/or fatty acetates. In certain embodiments, the short chain fatty alcohols, fatty aldehydes and/or fatty acetates have carbon chain length shorter than or equal to C18. In some preferred embodiments of methods to produce short chain pheromones, select enzymes which prefer to store short-chain fatty acyl-CoAs are co-expressed with one or more fatty acyl desaturase. Such suitable acyltransferase enzymes are exemplified by heterologous or engineered glycerol-3-phosphate acyl transferases (GPATs), lysophosphatidic acid acyltransferases (LPAATs), glycerolphospholipid acyltransferase (GPLATs) and/or diacylglycerol acyltransferases (DGATs). Examples of acyl transferases that are suitable for this purpose are listed in Table 5b.
In some preferred embodiments of methods to produce fatty alcohols, fatty aldehydes and/or fatty acetates, one or more genes of the microbial host encoding glycerol-3-phosphate acyl transferases (GPATs), lysophosphatidic acid acyltransferases (LPAATs), glycerolphospholipid acyltransferase (GPLATs) and/or diacylglycerol acyltransferases (DGATs) are deleted or downregulated, and replaced with one or more GPATs, LPAATs, GPLATs, or DGATs which prefer to store short-chain fatty acyl-CoAs. Such deletion or downregulation targets include but are not limited to Y. lipolytica YALI0C00209g, Y. lipolytica YALI0E18964g, Y. lipolytica YALI0F19514g, Y. lipolytica Y. lipolytica YALI0C14014g, Y. lipolytica YALI0E16797g, Y. lipolytica YALI0E32769g, Y. lipolytica YALI0D07986g, S. cerevisiae YBL011w, S. cerevisiae YDL052c, S. cerevisiae YOR175C, S. cerevisiae YPR139C, S. cerevisiae YNR008w, S. cerevisiae YOR245c, Candida 1503 02577, Candida CTRG_02630, Candida CaO19.250, Candida CaO19.7881, Candida CTRG_02437, Candida CaO19.1881, Candida CaO19.9437, Candida CTRG_01687, Candida CaO19.1043, Candida CaO19.8645, Candida CTRG_04750, Candida CaO19.13439, Candida CTRG_04390, Candida CaO19.6941, Candida CaO19.14203, and Candida CTRG_06209. In other embodiments, the acyltransferase is inserted at the AXP Acid extracellular protease locus (YALI0B05654g).
Thus, in some embodiments, the present disclosure teaches a recombinant microorganism comprising an acyltransferase exhibiting at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, or 50% sequence identity with any one of SEQ ID Nos. selected from the group consisting of 92.
Thus, in some embodiments, the present disclosure teaches a recombinant microorganism comprising a nucleic acid molecule encoding for an acyltransferase, wherein said nucleic acid molecule exhibits at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, or 50% sequence identity with any one of SEQ ID Nos. selected from the group consisting of 91.
In some embodiments, the present disclosure teaches a recombinant microorganism comprising at least one nucleic acid molecule encoding an acyltransferase having at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, or 50% sequence identity to an acyltransferase selected from the group consisting of SEQ ID NO: 92.
In some embodiments, the present disclosure teaches a recombinant microorganism comprising a deletion, disruption, mutation, and or reduction in the activity of one or more acyltransferases selected from the group consisting of YALI0E32791g (DGA1) and/or YALI0D07986g (DGA2).
Thus, in some embodiments, the recombinant microorganism of the present disclosure will comprise deletions or other disruptions in endogenous genes encoding proteins exhibiting at least 100%, 99%, 98%, 97%, 95%, 94%, 93%, 92%, 91%, or 90% sequence identity with the proteins encoded by YALI0E32791g (DGA1) and YALI0D07986g (DGA2).
Thus, in some embodiments, the recombinant microorganism of the present disclosure will comprise deletions in endogenous genes encoding proteins exhibiting at least 100%, 99%, 98%, 97%, 95%, 94%, 93%, 92%, 91%, or 90% sequence identity with uniprot database ID Q6C3R1 (DGA1) and/or Q6C9V5 (DGA2).
Glycerol-3-Phosphate Acyltransferase (GPAT)
The present disclosure describes enzymes that catalyze the acylation reaction at the sn-1 position of glycerol 3-phosphate shown as follows:
a long-chain acyl-CoA+sn-glycerol 3-phosphate→a 1-acyl-sn-glycerol 3-phosphate+coenzyme A.
Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the acylation reaction at the sn-1 position of glycerol 3-phosphate. The plant cell contains three types of GPAT, which are located in the chloroplasts, mitochondria and cytoplasm. The enzyme in chloroplasts is soluble and uses acyl-(acyl-carrier protein) as the acyl donor, whereas the enzymes in the mitochondria and the cytoplasm are bound to membranes and use acyl-CoA as the acyl donor (Nishida I et al. (1993) The gene and the RNA for the precursor to the plastid-located glycerol-3-phosphate acyltransferase of Arabidopsis thaliana. Plant Mol Biol. 21(2): 267-77; Murata N and Tasaka Y (1997) Glycerol-3-phosphate acyltransferase in plants. Biochim Biophys Acta. 1348(1-2):10-16).
Eight GPAT genes have been identified in Arabidopsis (Zheng Z et al. (2003) Arabidopsis AtGPAT1, a member of the membrane-bound glycerol-3-phosphate acyltransferase gene family, is essential for tapetum differentiation and male fertility. Plant Cell 15(8):1872-87). GPAT1 was shown to encode a mitochondrial enzyme (Zheng et al. 2003). GPAT4, GPAT5 and GPAT8 were shown to be essential for cutin biosynthesis (Beisson F et al. (2007) The acyltransferase GPATS is required for the synthesis of suberin in seed coat and root of Arabidopsis. Plant Cell 19(1): 351-368; Li, Y et al. (2007) Identification of acyltransferases required for cutin biosynthesis and production of cutin with suberin-like monomers. Proc Natl Acad Sci USA 104(46): 18339-18344). GPAT2, GPAT3, GPAT6 and GPAT7 have not been characterized yet.
The cytoplasmic GPAT is responsible for the synthesis of triacylglycerol and non-chloroplast membrane phospholipids. It is expected to have a substrate preference for palmitate (C16:0) and oleate (C18:1) since these two fatty acids are the most common ones found at the sn-1 position of plant triacylglycerols. The cytoplasmic GPAT was partially purified from avocado (Eccleston V S and Harwood J L (1995) Solubilisation, partial purification and properties of acyl-CoA: glycerol-3-phosphate acyltransferase from avocado (Persea americana) fruit mesocarp. Biochim Biophys Acta 1257(1):1-10).
Membrane-bound glycerol-3-phosphate acyltransferase (PlsB) from E. coli catalyzes the first committed step in phospholipid biosynthesis and is thought to function in close proximity to the succeeding enzyme 1-acylglycerol-3-phosphate O-acyltransferase (PlsC) (Kessels J M et al. (1983) Facilitated utilization of endogenously synthesized lysophosphatidic acid by 1-acylglycerophosphate acyltransferase from Escherichia coli. Biochim Biophys Acta 753(2): 227-235). It is specific for acylation at position 1 of sn-glycerol 3-phosphate and can utilize either fatty acyl-acyl carrier protein (acyl-ACP) or fatty acyl-coenzyme A (acyl-CoA) thioesters as acyl donors to form a 1-acyl-sn-glycerol 3-phosphate. Fatty acids that are endogenously synthesized are attached to ACP and exogenously added fatty acids are attached to CoA. In E. coli phospholipids, the sn 1 position is occupied mainly by either palmitate, or cis-vaccenate, whereas the sn 2 position is predominantly palmitoleate, or cis-vaccenate. This is thought to result from the substrate preferences of the PlsB and PlsC enzymes.
The plsB gene has been shown to be regulated by stress response regulators such as RNA polymerase, sigma 24 (sigma E) factor and ppGpp (Wahl A et al. (2011) Antagonistic regulation of dgkA and plsB genes of phospholipid synthesis by multiple stress responses in Escherichia coli. Mol Microbiol 80(5): 1260-75. PlsB is part of a protein network for phospholipid synthesis and interacts with a holo-[acyl-carrier protein] (ACP), esterase/thioesterase (YbgC) and phosphatidylserine synthase (PssA) to form a complex at the cytoplasmic side of the inner membrane.
plsB is essential for growth (Baba T et al. (2006) Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol. 2:2006-2008; Yoshimura M et al. (2007) Involvement of the YneS/YgiH and PlsX proteins in phospholipid biosynthesis in both Bacillus subtilis and Escherichia coli. BMC Microbiol 7: 69).
Site-directed mutagenesis and chemical modification studies have demonstrated catalytically important amino acid residues in PlsB, including an invariant histidine residue that is essential for catalysis (Lewin T M et al. (1999) Analysis of amino acid motifs diagnostic for the sn-glycerol-3-phosphate acyltransferase reaction. Biochemistry 38(18): 5764-5771). Genetic studies have identified the plsB locus as involved in the formation of multidrug tolerant persister cells.
The properties of the E. coli B enzyme were studied in earlier work (Kito M et al. (1972) Inhibition of L-glycerol 3-phosphate acyltransferase from Escherichia coli by cis-9,10-methylenehexadecanoic acid. J Biochem 71(1): 99-105; Okuyama H and Wakil S J (1973) Positional specificities of acyl coenzyme A: glycerophosphate and acyl coenzyme A: monoacylglycerophosphate acyltransferases in Escherichia coli. J Biol Chem 248(14): 5197-5205; Kito M et al. (1978) Function of phospholipids on the regulatory properties of solubilized and membrane-bound sn-glycerol-3-phosphate acyltransferase of Escherichia coli. Biochim Biophys Acta 529(2): 237-249).
A glycerol-3-phosphate/dihydroxyacetone phosphate dual substrate-specific sn-1 acyltransferase is located in lipid particles and the ER and is involved in the stepwise acylation of glycerol-3-phosphate and dihydroxyacetone in lipid biosynthesis. The most conserved motifs and functionally relevant residues are oriented towards the ER lumen.
A gene (SCT1) encoding a dual glycerol-3-phosphate O-acyltransferase (GAT)/dihydroxyacetone phosphate acyltransferase (DHAT) was identified, cloned and biochemically characterized from Saccharomyces cerevisiae. In the yeast Δgpt1 mutant which exhibits very low GAT/DHAT activity, the overexpression of SCT1 through a plasmid vector showed increased GAT/DHAT activity underlining the proposed molecular function as glycerol-3-phosphate O-acyltransferase/dihydroxyacetone phosphate acyltransferase. The GAT/DHAT activity towards acyl-donors was highest with palmitoleoyl-CoA followed by palmitoyl-CoA, oleoyl-CoA and stearoyl-CoA. The SCT1p was localized to membranes in the cytosol, most probably to the endoplasmic reticulum. In vivo studies of Δsct1 mutants did reveal an impact on all four phospholipids but the observed decrease of 16:0 fatty acids in the phosphatidylethanolamine class was balanced out by an increase in other fatty acids, particularly 18:0 molecular species. The null mutants of SCT1 and GPT2 were synthetically lethal in yeast (Zheng Z and Zou J (2001) The initial step of the glycolipid pathway: identification of glycerol 3-phosphate/dihydroxyacetone phosphate dual substrate acyltransferases in Saccharomyces cerevisiae. J Biol Chem 276(45): 417104-41716).
The gene (GPT2) encoding a dual glycerol-3-phosphate O-acyltransferase (GAT)/dihydroxyacetone phosphate acyltransferase (DHAT) from Saccharomyces cerevisiae was identified, cloned and biochemically characterized. GPT2 was recombinantly expressed in E. coli in the ΔplsB background devoid of GAT/DHAT activity and showed an increased GAT activity but could not rescue the mutant probably because of the incorrect embedding of GPT2 in the membrane. In the yeast Δgpt1 mutant which exhibits very low GAT/DHAT activity, the overexpression of GPT2 from a plasmid vector showed increased GAT/DHAT activity, underlining the proposed molecular function as glycerol-3-phosphate O-acyltransferase/dihydroxyacetone phosphate acyltransferase. The GAT/DHAT activity towards acyl-donors was highest with oleoyl-CoA followed by palmitoleoyl-CoA, palmitoyl-CoA and stearoyl-CoA.
The GPT2p was localized to membranes in the cytosol. In vivo studies of Δgpt2 mutants did not reveal any significant impact on the total fatty acid profile but a decrease of 16:1 fatty acids in the phosphatidylethanolamine class was observed which was compensated by an increase in 16:0 and 18:1 molecular species. Analysis of a known yeast mutant TTA1 deficient in GAT activity showed that the TTA1 GPT2 gene had a missense mutation with one nucleotide change in the conserved motif III for acyltransferases. The null mutants of SCT1 and GPT2 were synthetically lethal in yeast (Zheng and Zou 2001).
In some embodiments, the glycerol-3-phosphate acyltransferase is a GPAT from Arabidopsis thaliana (At1g02390). In some embodiments, the glycerol-3-phosphate acyltransferase is PlsB from E. coli (Gene ID EG10740). In some embodiments, the glycerol-3-phosphate acyltransferase is the dual glycerol-3-phosphate O-acyltransferase (GAT)/dihydroxyacetone phosphate acyltransferase (DHAT) SCT1 from S. cerevisiae (YBL011w). In some embodiments, the glycerol-3-phosphate acyltransferase is YALI0C00209g from Yarrowia lipolytica. In some embodiments, the glycerol-3-phosphate acyltransferase is 1503_02577 from Candida albicans. In some embodiments, the glycerol-3-phosphate acyltransferase is CTRL 02630 from Candida tropicalis. In some embodiments, the glycerol-3-phosphate acyltransferase is the dual glycerol-3-phosphate O-acyltransferase (GAT)/dihydroxyacetone phosphate acyltransferase (DHAT) GPT2 from S. cerevisiae (YKR067w). In some embodiments, the glycerol-3-phosphate acyltransferase is CaO19.5815 from Candida albicans. In some embodiments, the glycerol-3-phosphate acyltransferase is CaO19.13237 from Candida albicans. In some embodiments, the glycerol-3-phosphate acyltransferase is CTRL 02630 from Candida tropicalis.
Lysophosphatidic Acid Acyltransferase (LPAAT)
The present disclosure describes enzymes that catalyze acylation of the sn-2 position of triacylglycerol.
Membrane-bound 1-acylglycerol-3-phosphate O-acyltransferase encoded by gene plsC catalyzes the second step in phospholipid biosynthesis and is thought to function in close proximity to the preceding enzyme glycerol-3-phosphate acyltransferase encoded by gene plsB (Kessels J M et al. 1983). It is specific for acylation at the sn-2 position of a 1-acyl-sn-glycerol 3-phosphate and can utilize either acyl-acyl carrier protein (acyl-ACP), or acyl-coenzyme A (acyl-CoA) as the fatty acyl donor to form a 1,2-diacyl-sn-glycerol 3-phosphate (a phosphatidate, a phosphatidic acid). Fatty acids that are endogenously synthesized are attached to ACP and exogenously added fatty acids are attached to CoA (Greenway D L and Silbert D F (1983) Altered acyltransferase activity in Escherichia coli associated with mutations in acyl coenzyme A synthetase. J Biol Chem 258(21): 13034-13042). In E. coli phospholipids at the sn 1 position is occupied mainly by either palmitate, or cis-vaccenate, whereas the sn 2 position is predominantly palmitoleate, or cis-vaccenate. This is thought to result from the substrate preferences of the PlsB and PlsC enzymes (Rock C O et al. (1981) Phospholipid synthesis in Escherichia coli. Characteristics of fatty acid transfer from acyl-acyl carrier protein to sn-glycerol 3-phosphate. J Biol Chem 256(2): 736-742; Goelz S E and Cronan J E (1980) The positional distribution of fatty acids in Escherichia coli phospholipids is not regulated by sn-glycerol 3-phosphate levels. J Bacteriol 144(1): 462-464).
Site directed mutagenesis studies showed that changing threonine-122 to alanine or leucine resulted in changes in acyl-CoA substrate specificity (Morand L Z et al. (1998) Alteration of the fatty acid substrate specificity of lysophosphatidate acyltransferase by site-directed mutagenesis. Biochem Biophys Res Commun 244(1): 79-84).
In an engineered strain of E. coli, overexpression of PlsC and GalU resulted in increased production of glycoglycerolipids (Mora-Buye N et al. (2012). An engineered E. coli strain for the production of glycoglycerolipids. Metab Eng 14(5): 551-559).
The plsC gene of Streptococcus pneumoniae encodes a 1-acylglycerol-3-phosphate acyltransferase homologous to the E. coli enzyme. The gene was cloned and expressed in E. coli, and membranes expressing it were shown to catalyze the predicted function (Lu Y J et al. (2006) Acyl-phosphates initiate membrane phospholipid synthesis in Gram-positive pathogens. Mol Cell 23(5): 765-772).
Plant lysophosphatidate acyltransferase (LPAAT) catalyzes acylation of the sn-2 position of triacylglycerol. The substrate specificity of LPAAT in a given plant species generally determines what fatty acid species are incorporated at the sn-2 position. LPAAT has been cloned from maize and meadow foam. There are two LPAAT genes in meadow foam, whereas only one in maize. The enzyme activity of both LAT1 and LAT2 in meadow foam was confirmed by in vitro assay. In addition, LAT2 was shown to functional complement the E. coli LPAAT deficient strain (Brown A P et al. (2002) Limnanthes douglasii lysophosphatidic acid acyltransferases: immunological quantification, acyl selectivity and functional replacement of the Escherichia coli plsC gene. Biochem J 364(Pt 3):795-805).
LAT1 is a highly selective acyltransferase that only uses 18:1-CoA as a substrate. LAT2 is less selective. The highest activity was shown towards 22:1-CoA, followed by 16:0- and 18:1-CoA. The substrate specificities of LAT1 and LAT2 are consistent with their proposed roles, for LAT1 in membrane lipid biosynthesis and LAT2 in storage lipid biosynthesis. Plant cell membranes predominantly contain C16 and C18 unsaturated fatty acids, whereas storage lipids contain a wide range of fatty acids including saturated fatty acids and very long-chain unsaturated fatty acids. The protein level of LAT1 and LAT2 in different plant tissues was detected by antibodies. LAT1 is present in both leaves and developing seeds, whereas LAT2 is only detected in developing seeds. This again is consistent with their proposed roles. The role of LAT2 in triacylglycerol biosynthesis was further shown by transformation of LAT2 in oil seed rape which does not normally contain 22:1-CoA at the sn-2 position. The transformation of the meadow foam LAT2 inserted 22:1-CoA at the sn-2 position (Lassner M W et al. (1995) Lysophosphatidic acid acyltransferase from meadowfoam mediates insertion of erucic acid at the sn-2 position of triacylglycerol in transgenic rapeseed oil. Plant Physiol 109(4): 1389-1394).
Utilizing viable mutant Saccharomyces cerevisiae strains lacking sphingolipid biosynthesis, the gene SLC1 was isolated and demonstrated to encode an acyl-CoA: lysophosphatidate acyltransferase. Sequence homology with the PLSC protein of E. coli which is classified as 1-acyl-sn-glycerol-3-phosphate acyltransferase indicated a similar function. This presumed molecular function of SLC1p was corroborated by the ability to rescue the ΔplsC mutant of E. coli. It could be shown that a single nucleotide alteration changing an L-glutamine to an L-leucine at position 131 transformed the substrate preference from C16 and C18 fatty acids to C26 fatty acids which was reflected in vivo in the corresponding fatty acid composition of wild type (SLC1) versus mutant (SLC1-1) (Nagiec M M et al. (1993) A suppressor gene that enables Saccharomyces cerevisiae to grow without making sphingolipids encodes a protein that resembles an Escherichia coli fatty acyltransferase. J Biol Chem 268(29): 22156-22163).
In vitro assays with the recombinantly expressed and purified SLC1p in E. coli revealed a substrate preference towards lyso-phosphatidate and oleoyl-CoA but also accepted 1-palmitoylglycerol 3-phosphate and 1-stearoyl-sn-glycerol 3-phosphate. In vivo studies of mutants such as Δslc1, Δslc4 (another potential acyl-CoA:phosphatidyl acyltransferase) and double mutants of Δslc1Δslc4 bearing a plasmid with either the SLC1 or SLC4 gene referred to as 2.ΔSLC1 (or 2.ΔSLC4) showed that SLC1 promoted the biosyntheses of phosphatidate and also phosphatidylinositol and diacylglycerol. It was suggested that SLC1 is involved in phospholipid remodeling by exchanging fatty acids on glycerophospholipids in vivo (Benghezal M et al. (2007) SLC1 and SLC4 encode partially redundant acyl-coenzyme A 1-acylglycerol-3-phosphate O-acyltransferases of budding yeast. J Biol Chem 282(42): 30845-30855).
Screening the yeast genome with candidate open reading frames (ORFs) of known acyltransferase enzymes and testing the associated deletion strains, the gene encoding an acyl-CoA dependent lyso-phospholipid acyltransferase (ALE1) was identified. In the Δale1 strain a dramatic decrease of lyso-phosphatidylethanolamine acyltransferase (LPEAT) activity was observed but it could also be demonstrated that ALE1p may provide redundant lyso-phosphatidate acyltransferase (LPAAT) activity when the main LPAAT in Saccharomyces cerevisiae, i.e. SLC1p, is absent or rendered inactive. ALE1p preferably attaches unsaturated acyl chains of varying length to the sn-2 position of lyso-phospholipids. The enzyme was localized to both microsomal and mitochondrial membranes utilizing high purity cell fractionation. It has been proposed that ALE1 may be the major LPEAT in the exogenous lysolipid metabolism (ELM) pathway in yeast but it is also required for efficient functioning of the endogenous Kennedy pathway (Riekhof W R et al. (2007) Identification and characterization of the major lysophosphatidylethanolamine acyltransferase in Saccharomyces cerevisiae. J Biol Chem 282(39): 28344-28352).
In a simultaneous study, LPT1 (synonymous to ALE1) was identified by applying a synthetic genetic array analysis and shown to have lyso-phospholipid acyltransferase activity. In this study the best substrate for LPT1 (=ALE1) was lyso-phosphatidylcholine, hence acting as a lyso-phosphatidylcholine acyltransferase (LPCAT) and the residual activity as LPAAT reported earlier was also demonstrated utilizing single Δlpt1 and double Δscl1Δlpt1 mutants, the latter being inviable. The ratio of incorporating oleate into phosphatidylcholine was determined as 70% towards the de novo synthesis and 30% towards remodeling (Jain S et al. (2007) Identification of a novel lysophospholipid acyltransferase in Saccharomyces cerevisiae. J Biol Chem 282(42): 30562-30569).
The molecular function of ALE1 (also referred to as LCA1 or SLC4) as a lyso-phosphatidylcholine acyltransferase (LPCAT) was corroborated in another simultaneous study monitoring the incorporation of radioactive labeled lyso-phosphatidylcholine and/or palmitoyl-CoA into phosphatidylcholine (PC). The study confirmed that ALE1p (=LCA1p in this study) was accepting a variety of acyl-donors but showed highest activity as LPCAT regardless of the acyl-chain of lyso-phosphatidylcholine species (16:0 or 18:1). In addition, a high sensitivity towards Zn2+ was observed which was inhibitory at concentrations above 0.1 mM and activating at lower concentrations (10 to 25 μM). The high PC turnover-rate measured for ALE1p (=LCA1p) emphasized the enzyme as a key catalyst involved in the re-acylation of PC (Chen Q et al. (2007) The yeast acylglycerol acyltransferase LCA1 is a key component of Lands cycle for phosphatidylcholine turnover.” FEBS Lett 581(28): 5511-5516).
The search for genes causing aberrations in the formation of lipid droplets (LD) in Saccharomyces cerevisiae identified the gene LOA1 (formerly VPS66) encoding for an acyl-CoA dependent lysophosphatidate acyltransferase. The in vivo molecular function of LOA1p was determined using the comparison of the lipidome of wild type and Δloa1 yeast strains. The analysis showed that in the LOA1 deficient mutant (Δloa1) the percentage of oleate containing phosphatidate molecular species was considerably reduced and the content of triacylglycerols (TGA) was lowered by 20 percent. The protein was recombinantly expressed in E. coli and partially purified by obtaining the highly enriched lipid droplet fraction and by affinity chromatography with LOA1p still attached to the matrix beads. The purified LOA1p was characterized in in vitro assays demonstrating that LOA1p was specific for lysophosphatidate and oleoyl-CoA, thus acting as a oleoyl-CoA: lysophosphatidate acyltransferase in yeast. Based upon the results, LOA1p was proposed to be significantly involved in channeling excess oleate-containing phosphatidate species into TAG biosynthesis and the proper development of lipid droplets (LD's). Utilizing a genomic-tagging construct, subcellular fractionation, immunohistochemistry and fluorescence microscopy LOA1 could be localized to both endoplasmic reticulum (ER) and lipid droplets (LD's) (Ayciriex S et al. (2012) YPR139c/LOA1 encodes a novel lysophosphatidic acid acyltransferase associated with lipid droplets and involved in TAG homeostasis. Mol Biol Cell 23(2): 233-246).
In some embodiments, the lysophosphatidic acid acyltransferase is plsC from E. coli (MetaCyc Accession ID EG11377). In other embodiments, the lysophosphatidic acid acyltransferase is plsC from S. pneumoniae (MetaCyc Accession ID G-10763). In some embodiments, the lysophosphatidic acid acyltransferase is LAT1 from Limnanthes douglasii. In some embodiments, the lysophosphatidic acid acyltransferase is LAT2 from Limnanthes douglasii (MetaCyc Accession ID G-9398). In some embodiments, the lysophosphatidic acid acyltransferase is SLC1 from Saccharomyces cerevisiae (YDL052c). In some embodiments, the lysophosphatidic acid acyltransferase is YALI0E18964g from Yarrowia lipolytica. In some embodiments, the lysophosphatidic acid acyltransferase is CaO19.250 from Candida albicans. In some embodiments, the lysophosphatidic acid acyltransferase is CaO19.7881 from Candida albicans. In some embodiments, the lysophosphatidic acid acyltransferase is CTRL_02437 from Candida tropicalis. In some embodiments, the lysophosphatidic acid acyltransferase is ALE1 from Saccharomyces cerevisiae (YOR175C). In some embodiments, the lysophosphatidic acid acyltransferase is YALI0F19514g from Yarrowia lipolytica. In some embodiments, the lysophosphatidic acid acyltransferase is CaO19.1881 from Candida albicans. In some embodiments, the lysophosphatidic acid acyltransferase is CaO19.9437 from Candida albicans. In some embodiments, the lysophosphatidic acid acyltransferase is CTRL_01687 from Candida tropicalis. In some embodiments, the lysophosphatidic acid acyltransferase is LOA1 from Saccharomyces cerevisiae (YPR139C). In some embodiments, the lysophosphatidic acid acyltransferase is YALI0C14014g from Yarrowia lipolytica. In some embodiments, the lysophosphatidic acid acyltransferase is CaO19.1043 from Candida albicans. In some embodiments, the lysophosphatidic acid acyltransferase is CaO19.8645 from Candida albicans. In some embodiments, the lysophosphatidic acid acyltransferase is CTRL_04750 from Candida tropicalis.
Glycerolphospholipid Acyltransferase (GPLAT)
The present disclosure describes enzymes that catalyze the following reaction:
1-alkyl-sn-glycero-3-phosphoethanolamine+a 2-acyl-1-alkyl-sn-glycero-3-phosphocholine an O-1-alkyl-2-acyl-sn-glycero-3-phosphoethanolamine+a 1-alkyl-2-lyso-sn-glycero-3-phosphocholine
GPLAT enzymes catalyze the transfer of fatty acids from intact choline- or ethanolamine-containing glycerolphospholipids to the sn-2 position of a lyso-glycerolphospholipid. The organyl group on sn-1 of the donor or acceptor molecule can be alkyl, acyl or alk-1-enyl. The term ‘radyl’ has sometimes been used to refer to such substituting groups. The enzyme requires Coenzyme A and does not favor the transfer of polyunsaturated acyl groups.
Diacylglycerol Acyltransferase (DGAT)
The present disclosure describes enzymes that add an acyl group to the sn-3 position of diacylglycerol (DAG) to form triacylglycerol (TAG).
Diacylglycerol acyltransferase (DGAT) catalyzes the only unique reaction in triacylglycerol biosynthesis. It adds an acyl group to the sn-3 position of diacylglycerol (DAG) and forms triacylglycerol (TAG), shown as follows:
an acyl-CoA+a 1,2-diacyl-sn-glycerol→a triacyl-sn-glycerol+coenzyme A.
DGAT accepts a broad range of acyl-CoA as acyl donor including C18:1, C18:2, and C20:1 acyl-CoA as demonstrated for the Arabidopsis DGAT (Jako C et al. (2001) Seed-specific over-expression of an Arabidopsis cDNA encoding a diacylglycerol acyltransferase enhances seed oil content and seed weight. Plant Physiol 126(2): 861-874). Expressing the Arabidopsis cDNA of DGAT in an insect cell culture and in yeast, as well as over-expressing the cDNA in wild type Arabidopsis, demonstrated the DGAT activity in transferring an acyl group to the sn-3 position of DAG (Hobbs D H et al. (1999) Cloning of a cDNA encoding diacylglycerol acyltransferase from Arabidopsis thaliana and its functional expression. FEBS Lett 452(3): 145-149; Zou J et al. (1999) The Arabidopsis thaliana TAG1 mutant has a mutation in a diacylglycerol acyltransferase gene. Plant J 19(6): 645-653). Over-expression of the Arabidopsis cDNA in wild type Arabidopsis increased oil deposition in seeds and this increase is correlated to the increased mRNA expression level of DGAT. This indicates that DGAT is a regulatory point of the triacylglycerol biosynthesis pathway.
The gene encoding the bifunctional acyl-CoA:acylglycerol acyltransferase (DGAT) has been identified in Saccharomyces cerevisiae as a major contributor to triacylglycerol biosynthesis (Sandager L et al. (2002) Storage lipid synthesis is non-essential in yeast. J Biol Chem 277(8): 6478-6482). The gene (DGA1) belongs in the DGAT2 family which members are characterized as acyl-CoA dependent acyltransferases (Lardizabal K D et al. (2001) DGAT2 is a new diacylglycerol acyltransferase gene family: purification, cloning, and expression in insect cells of two polypeptides from Mortierella ramanniana with diacylglycerol acyltransferase activity.” J Biol Chem 276(42): 38862-38869). It has been demonstrated that DGA1p is the only acyl-CoA dependent acyltransferase catalyzing the esterification of diacylglycerol (DAG) to triacylglycerol (TAG) in the yeast genome. This has been shown in deletion mutants of DGA1 (Δdga1) and in combination with the deletion of the other diacylglycerol acyltransferase of importance in yeast, i.e. LRO1 which esterifies DAG utilizing a phospholipid acyl donor (Δlro1). In the Δdga1Δlro1 double mutant almost all of the diacylglycerol acyltransferase has been lost and TAG synthesis was abolished. A plasmid carrying the DGA1 gene could rescue the TAG synthetic deficiency in the mutant indicating that in vivo DGA1 was prominently involved in the TAG biosynthetic route (Sorger D, Daum G (2002). Synthesis of triacylglycerols by the acyl-coenzyme A:diacylglycerol acyltransferase Dga1p in lipid particles of the yeast Saccharomyces cerevisiae. J Bacteriol 184(2): 519-524; Oelkers P et al. (2002) The DGA1 gene determines a second triglyceride synthetic pathway in yeast. J Biol Chem 277(11): 8877-8881). In vitro a preference of DGA1p towards oleoyl-CoA and palmitoyl-CoA was observed which is inverted for the phospholipid dependent acyltransferase LRO1p (Oelkers et al. 2002).
In addition, the function of DGA1p as an acyl-CoA dependent monoacylglycerol acyltransferase (MGAT) was demonstrated in vivo utilizing Δdga1 mutants which had lost more than 60% of the MGAT activity. The in vitro MGAT activity of DGA1 was shown by the oleoyl-CoA dependent esterification of 2-oleoylglycerol yielding 1,2-dioleoylglycerol in the process (Heier C et al. (2010) Identification of Yju3p as functional orthologue of mammalian monoglyceride lipase in the yeast Saccharomyces cerevisiae. Biochim Biophys Acta 1801(9): 1063-1071).
More insights into the functional importance and topological orientation of sequence motifs in the primary sequence of DGA1p has been gained by in silico analyses, site-directed mutagenesis of signature motifs and deletion mutations of the C- and N-termini. It could be demonstrated that besides the signature motifs found in other DGAT2 family members Saccharomyces possesses a unique hydrophilic stretch which was shown to significantly modulate enzyme activity. Also, the histidine residue 195 in the second of the four determined transmembrane domains was proven to be essential for enzyme activity. The topology of DGA1 revealed that both C- and N-termini face the cytoplasm and that the C-terminus was more important for DGA1 activity than the N-terminus (Liu Q et al. (2011) Functional and topological analysis of yeast acyl-CoA:diacylglycerol acyltransferase 2, an endoplasmic reticulum enzyme essential for triacylglycerol biosynthesis. J Biol Chem 286(15): 13115-13126).
Using highly purified cell fragments and immunoblotting, Sorger et al. (2002) and Liu et al. (2011) demonstrated that DGA1 was localized to lipid droplets and microsomal membranes, most probably the endoplasmic reticulum.
Acinetobacter sp. ADP1 expresses a bifunctional enzyme that exhibits both wax ester synthase (WS) and acyl-coA:diacylglycerol acyltransferase (DGAT) activities (Kalscheuer R and Steinbuchel A (2003) A novel bifunctional wax ester synthase/acyl-CoA:diacylglycerol acyltransferase mediates wax ester and triacylglycerol biosynthesis in Acinetobacter calcoaceticus ADP1. J Biol Chem 278(10): 8075-8082). This homodimer catalyzes the final steps in TAG and WE biosynthesis (Stoveken T et al. (2005) The wax ester synthase/acyl coenzyme A:diacylglycerol acyltransferase from Acinetobacter sp. strain ADP1: characterization of a novel type of acyltransferase. J Bacteriol 187(4): 1369-1376). It mediates both oxo ester and thio ester bond formation and has a broad substrate range, accepting medium chain fatty alcohols and acyl-CoA esters as well as monoacylglycerols (MAGs) (Uthoff S et al. (2005) Thio wax ester biosynthesis utilizing the unspecific bifunctional wax ester synthase/acyl coenzyme A:diacylglycerol acyltransferase of Acinetobacter sp. strain ADP1. Appl Environ Microbiol 71(2): 790-796).
In some embodiments, the diacylglycerol acyltransferase is TAG1 from Arabidopsis thaliana (Gene ID AT2G19450). In some embodiments, the diacylglycerol acyltransferase is DGA1 from S. cerevisiae (YOR245c). In some embodiments, the diacylglycerol acyltransferase is atfA from Acinetobacter sp. ADP1 (MetaCyc Accession ID ACIAD0832). In some embodiments, the diacylglycerol acyltransferase is YALI0E32769g from Yarrowia lipolytica. In some embodiments, the diacylglycerol acyltransferase is CaO19.6941 from Candida albicans. In some embodiments, the diacylglycerol acyltransferase is CaO19.14203 from Candida albicans. In some embodiments, the diacylglycerol acyltransferase is CTRL 06209 from Candida tropicalis.
Phospholipid: diacylglycerol acyltransferase (PDAT) catalyzes the following reaction: a phosphatidylcholine+a 1,2-diacyl-sn-glycerol→a triacyl-sn-glycerol+a 1-acyl-sn-glycero-3-phosphocholine.
The Arabidopsis PDAT can use different phospholipids as acyl donor, with acyl groups of 10-22 carbon chain length at either sn-positions (Stahl U et al. (2004) Cloning and functional characterization of a phospholipid: diacylglycerol acyltransferase from Arabidopsis. Plant Physiol 135(3):1324-1335). Acyl group at the sn-2 position of phosphatidylcholine is however used three times greater than at the sn-1 position. The highest activity is with acyl groups having multiple double bonds, epoxy or hydroxy groups. Among the tested, the enzyme activity was highest with ricinoleoyl. 18:0- and 22:1-acyl groups gave the lowest enzyme activity. Among different phospholipid species, higher activity is with phosphatidylethanolamine than with phosphatidate or phosphatidylcholine.
A PDAT activity was detected in castor bean seed microsome fraction. Radio-labeled ricinoleoyl and vernoloyl groups are effectively transferred from phosphatidylcholine to DAG forming triacylglycerol (Dahlqvist A et al. (2000) Phospholipid: diacylglycerol acyltransferase: an enzyme that catalyzes the acyl-CoA-independent formation of triacylglycerol in yeast and plants. Proc Natl Acad Sci USA 97(12): 6487-6492).
In other embodiments, the diacylglycerol acyltransferase is a phospholipid: diacylglycerol acyltransferase (PDAT). In some embodiments, the PDAT is from Arabidopsis thaliana (Gene ID AT5G13640). In some embodiments, the PDAT is from Ricinus communis. In some embodiments, the PDAT is LRO1 from Saccharomyces cerevisiae (YNR008w). In some embodiments, the PDAT is YALI0E16797g from Yarrowia lipolytica. In some embodiments, the PDAT is CaO19.13439 from Candida albicans. In some embodiments, the PDAT is CTRG_04390 from Candida tropicalis.
In some embodiments, a recombinant microorganism capable of producing a mono- or poly-unsaturated ≤C18 fatty alcohol, fatty aldehyde and/or fatty acetate from an endogenous or exogenous source of saturated C6-C24 fatty acid is provided, wherein the recombinant microorganism expresses one or more acyltransferase enzymes, and wherein the recombinant microorganism is manipulated to delete, disrupt, mutate, and/or reduce the activity of one or more endogenous acyltransferase enzymes. In some embodiments, the one or more acyltransferase enzymes being expressed are different from the one or more endogenous acyltransferase enzymes being deleted or downregulated. In some embodiments, the one or more endogenous or exogenous acyltransferase enzymes comprise glycerol-3-phosphate acyl transferases (GPATs), lysophosphatidic acid acyltransferases (LPAATs), glycerolphospholipid acyltransferase (GPLATs) and/or diacylglycerol acyltransferases (DGATs). In some embodiments, the one or more acyltransferase enzymes being expressed prefer to store short-chain fatty acyl-CoAs. In other embodiments, the one or more acyltransferase enzymes being expressed are selected from Table 5b. In some embodiments, the one or more endogenous acyltransferase enzymes being deleted or downregulated are selected from Y. lipolytica YALI0C00209g, Y. lipolytica YALI0E18964g, Y. lipolytica YALI0F19514g, Y. lipolytica YALI0C14014g, Y. lipolytica YALI0E16797g, Y. lipolytica YALI0E32769g, Y. lipolytica YALI0D07986g, S. cerevisiae YBL011w, S. cerevisiae YDL052c, S. cerevisiae YOR175C, S. cerevisiae YPR139C, S. cerevisiae YNR008w, S. cerevisiae YOR245c, Candida 1503 02577, Candida CTRG_02630, Candida CaO19.250, Candida CaO19.7881, Candida CTRG_02437, Candida CaO19.1881, Candida CaO19.9437, Candida CTRG_01687, Candida CaO19.1043, Candida CaO19.8645, Candida CTRG_04750, Candida CaO19.13439, Candida CTRG_04390, Candida CaO19.6941, Candida CaO19.14203, and Candida CTRG_06209. In some embodiments, the recombinant microorganism further expresses pheromone biosynthetic pathway enzymes. In further embodiments, the pheromone biosynthetic pathway enzymes comprise one or more fatty acyl desaturase and/or fatty acyl conjugase. In yet further embodiments, the pheromone biosynthetic pathway enzymes comprise one or more fatty alcohol forming fatty acyl reductase.
In some embodiments, a method of producing a mono- or poly-unsaturated ≤C18 fatty alcohol, fatty aldehyde and/or fatty acetate from an endogenous or exogenous source of saturated C6-C24 fatty acid are provided, wherein the method comprises introducing into or expressing in a recombinant microorganism at least one endogenous or exogenous nucleic acid molecule encoding an acyltransferase and introducing a deletion, insertion, or loss of function mutation in one or more gene encoding an acyltransferase, wherein the at least one endogenous or exogenous nucleic acid molecule encoding an acyltransferase being introduced or expressed is different from the one or more gene encoding an acyltransferase being deleted or downregulated. In some embodiments, the at least one endogenous or exogenous nucleic acid molecule encoding an acyltransferase being introduced or expressed or the one or more gene encoding an acyltransferase being deleted or downregulated comprise glycerol-3-phosphate acyl transferases (GPATs), lysophosphatidic acid acyltransferases (LPAATs), glycerolphospholipid acyltransferase (GPLATs) and/or diacylglycerol acyltransferases (DGATs). In some embodiments, the at least one endogenous or exogenous nucleic acid molecule encodes an acyltransferase that prefers to store short-chain fatty acyl-CoAs. In some embodiments, the at least one endogenous or exogenous nucleic acid molecule encodes an acyltransferase selected from Table 5b. In some embodiments, the one or more endogenous acyltransferase enzymes being deleted or downregulated are selected from Y. lipolytica YALI0C00209g, Y. lipolytica YALI0E18964g, Y. lipolytica YALI0F19514g, Y. lipolytica YALI0C14014g, Y. lipolytica YALI0E16797g, Y. lipolytica YALI0E32769g, Y. lipolytica YALI0D07986g, S. cerevisiae YBL011w, S. cerevisiae YDL052c, S. cerevisiae YOR175C, S. cerevisiae YPR139C, S. cerevisiae YNR008w, S. cerevisiae YOR245c, Candida 1503_02577, Candida CTRG_02630, Candida CaO19.250, Candida CaO19.7881, Candida CTRG_02437, Candida CaO19.1881, Candida CaO19.9437, Candida CTRG_01687, Candida CaO19.1043, Candida CaO19.8645, Candida CTRG_04750, Candida CaO19.13439, Candida CTRG_04390, Candida CaO19.6941, Candida CaO19.14203, and Candida CTRG_06209. In some embodiments, the method further comprises introducing into or expressing in the recombinant microorganism at least one endogenous or exogenous nucleic acid molecule encoding a fatty acyl desaturase and/or fatty acyl conjugase. In further embodiments, the method further comprises introducing into or expressing in the recombinant microorganism at least one endogenous or exogenous nucleic acid molecule encoding a fatty alcohol forming fatty acyl reductase.
Acylglycerol Lipases and Sterol Esterases
In some embodiments, recombinant microorganisms and methods are provided for the production of short chain fatty alcohols, fatty aldehydes and/or fatty acetates. In certain embodiments, the short chain fatty alcohols, fatty aldehydes and/or fatty acetates have carbon chain length shorter than or equal to C18. In some preferred embodiments of methods to produce short chain pheromones, select enzymes which prefer to hydrolyze ester bonds of long-chain acylglycerols are co-expressed with one or more fatty acyl desaturases. Such suitable enzymes are exemplified by heterologous or engineered acylglycerol lipases. Examples of acylglycerol lipases that are suitable for this purpose are listed in Table 5c.
In some preferred embodiments of methods to produce fatty alcohols, fatty aldehydes and/or fatty acetates, one or more genes of the microbial host encoding acylglycerol lipases (mono-, di-, or triacylglycerol lipases) and sterol ester esterases are deleted or downregulated and replaced with one or more acylglycerol lipases which prefer long chain acylglycerol substrates. Such deletion or down-regulation targets include, but are not limited to Y. lipolytica YALI0E32035g, Y. lipolytica YALI0D17534g, Y. lipolytica YALI0F10010g, Y. lipolytica YALI0C14520g, Y. lipolytica YALI0E00528g, S. cerevisiae YKL140w, S. cerevisiae YMR313c, S. cerevisiae YKR089c, S. cerevisiae YOR081c, S. cerevisiae YKL094W, S. cerevisiae YLL012W, S. cerevisiae YLR020C, Candida CaO19.2050, Candida CaO19.9598, Candida CTRG_01138, Candida W5Q_03398, Candida CTRG_00057, Candida CaO19.5426, Candida CaO19.12881, Candida CTRG_06185, Candida CaO19.4864, Candida CaO19.12328, Candida CTRG_03360, Candida CaO19.6501, Candida CaO19.13854, Candida CTRG_05049, Candida CaO19.1887, Candida CaO19.9443, Candida CTRG_01683, and Candida CTRG_04630.
Carboxylic ester hydrolases (EC 3.1.1) are a large class of enzymes catalyzing the hydrolysis or synthesis of ester bonds. They have been described in all life domains, prokaryotic and eukaryotic. Most of them belong to the α/β-hydrolase superfamily and have a conserved “catalytic triad” formed by His, an acidic amino acid and a Ser residue that is located in a highly conserved GXSXG sequence. During hydrolysis, the catalytic Ser will start the nucleophilic attack of the substrate helped by the other two residues from the triad, which are in close spatial vicinity. These are presumed to facilitate the hydrolysis of esters by a mechanism similar to that of chymotrypsin-like serine proteases. Another characteristic feature is the presence of an amino acidic region whose sequence is not as conserved as that of the catalytic triad, the oxyanion hole, which serves to stabilize a transition state generated during catalysis. In addition, these enzymes generally do not require cofactors. Acylglycerol lipases and sterol esterases belong to the carboxylic ester hydrolase family.
An acylglycerol lipase enzyme catalyzes a chemical reaction that uses water molecules to break the glycerol monoesters of long-chain fatty acids. The systematic name of this enzyme class is glycerol-ester acylhydrolase. Other names in common use include monoacylglycerol lipase, monoacylglycerolipase, monoglyceride lipase, monoglyceride hydrolase, fatty acyl monoester lipase, monoacylglycerol hydrolase, monoglyceridyllipase, and monoglyceridase. This enzyme participates in glycerolipid metabolism.
A sterol esterase enzyme catalyzes the chemical reaction:
steryl ester+H2O⇄sterol+fatty acid
Thus, the two substrates of this enzyme are steryl ester and H2O, whereas its two products are sterol and fatty acid.
The systematic name of this enzyme class is steryl-ester acylhydrolase. Other names in common use include cholesterol esterase, cholesteryl ester synthase, triterpenol esterase, cholesteryl esterase, cholesteryl ester hydrolase, sterol ester hydrolase, cholesterol ester hydrolase, cholesterase, and acylcholesterol lipase. This enzyme participates in bile acid biosynthesis. Sterol esterases are widespread in nature and have been identified from mammals' tissues such as the pancreas, intestinal mucosa, liver, placenta, aorta, and brain, to filamentous fungi, yeast, and bacteria.
In terms of substrate specificity, many sterol esterases are able to catalyze the hydrolysis or synthesis of a rather broad range of other substrates containing ester linkages, such as acylglycerols, aryl esters, and in some cases alcohol esters, cinnamyl esters, xhantophyl esters, or synthetic polymers.
In some embodiments, a recombinant microorganism capable of producing a mono- or poly-unsaturated ≤C18 fatty alcohol, fatty aldehyde and/or fatty acetate from an endogenous or exogenous source of saturated C6-C24 fatty acid is provided, wherein the recombinant microorganism expresses one or more acylglycerol lipase and/or sterol ester esterase enzymes, and wherein the recombinant microorganism is manipulated to delete, disrupt, mutate, and/or reduce the activity of one or more endogenous acylglycerol lipase and/or sterol ester esterase enzymes. In some embodiments, the one or more acylglycerol lipase and/or sterol ester esterase enzymes being expressed are different from the one or more endogenous acylglycerol lipase and/or sterol ester esterase enzymes being deleted or downregulated. In some embodiments, the one or more endogenous or exogenous acylglycerol lipase and/or sterol ester esterase enzymes being expressed prefer to hydrolyze ester bonds of long-chain acylglycerols. In other embodiments, the one or more acylglycerol lipase and/or sterol ester esterase enzymes being expressed are selected from Table 5c. In some embodiments, the one or more endogenous acylglycerol lipase and/or sterol ester esterase enzymes being deleted or downregulated are selected from Y. lipolytica YALI0E32035g, Y. lipolytica YALI0D17534g, Y. lipolytica YALI0F10010g, Y. lipolytica YALI0C14520g, Y. lipolytica YALI0E00528g, S. cerevisiae YKL140w, S. cerevisiae YMR313c, S. cerevisiae YKR089c, S. cerevisiae YOR081c, S. cerevisiae YKL094W, S. cerevisiae YLL012W, S. cerevisiae YLR020C, Candida CaO19.2050, Candida CaO19.9598, Candida CTRG_01138, Candida W5Q_03398, Candida CTRG_00057, Candida CaO19.5426, Candida CaO19.12881, Candida CTRG_06185, Candida CaO19.4864, Candida CaO19.12328, Candida CTRG_03360, Candida CaO19.6501, Candida CaO19.13854, Candida CTRG_05049, Candida CaO19.1887, Candida CaO19.9443, Candida CTRG_01683, and Candida CTRG_04630. In some embodiments, the recombinant microorganism further expresses pheromone biosynthetic pathway enzymes. In further embodiments, the pheromone biosynthetic pathway enzymes comprise one or more fatty acyl desaturase and/or fatty acyl conjugase. In yet further embodiments, the pheromone biosynthetic pathway enzymes comprise one or more fatty alcohol forming fatty acyl reductase.
In some embodiments, a method of producing a mono- or poly-unsaturated ≤C18 fatty alcohol, fatty aldehyde and/or fatty acetate from an endogenous or exogenous source of saturated C6-C24 fatty acyl-CoA are provided, wherein the method comprises introducing into or expressing in a recombinant microorganism at least one endogenous or exogenous nucleic acid molecule encoding an acylglycerol lipase or sterol ester esterase and introducing a deletion, insertion, or loss of function mutation in one or more gene encoding an acylglycerol lipase or sterol ester esterase, wherein the at least one endogenous or exogenous nucleic acid molecule encoding an acylglycerol lipase or sterol ester esterase being introduced or expressed is different from the one or more gene encoding an acylglycerol lipase or sterol ester esterase being deleted or downregulated. In some embodiments, the at least one endogenous or exogenous nucleic acid molecule encoding an acylglycerol lipase or sterol ester esterase being introduced or expressed prefers to hydrolyze ester bonds of long-chain acylglycerols. In some embodiments, the at least one endogenous or exogenous nucleic acid molecule encoding an acylglycerol lipase or sterol ester esterase being introduced or expressed is selected from Table 5c. In some embodiments, the one or more gene being deleted or downregulated encodes an acylglycerol lipase or sterol ester esterase selected from Y. lipolytica YALI0E32035g, Y. lipolytica YALI0D17534g, Y. lipolytica YALI0F10010g, Y. lipolytica YALI0C14520g, Y. lipolytica YALI0E00528g, S. cerevisiae YKL140w, S. cerevisiae YMR313c, S. cerevisiae YKR089c, S. cerevisiae YOR081c, S. cerevisiae YKL094W, S. cerevisiae YLL012W, S. cerevisiae YLR020C, Candida CaO19.2050, Candida CaO19.9598, Candida CTRG_01138, Candida W5Q_03398, Candida CTRG_00057, Candida CaO19.5426, Candida CaO19.12881, Candida CTRG_06185, Candida CaO19.4864, Candida CaO19.12328, Candida CTRG_03360, Candida CaO19.6501, Candida CaO19.13854, Candida CTRG_05049, Candida CaO19.1887, Candida CaO19.9443, Candida CTRG_01683, and Candida CTRG_04630. In some embodiments, the method further comprises introducing into or expressing in the recombinant microorganism at least one endogenous or exogenous nucleic acid molecule encoding a fatty acyl desaturase and/or fatty acyl conjugase. In further embodiments, the method further comprises introducing into or expressing in the recombinant microorganism at least one endogenous or exogenous nucleic acid molecule encoding a fatty alcohol forming fatty acyl reductase.
Expression of Toxic Proteins or Polypeptides
The present disclosure describes a toxic protein, peptide, or small molecule that can be encoded by a recombinant microorganism. In some embodiments, the toxic protein, peptide, or small molecule is biosynthetically produced along with an insect pheromone.
In some embodiments, the recombinant microorganism expresses one or more nucleic acid molecules encoding a protein or polypeptide which is toxic to an insect. In some embodiments, the toxic protein or polypeptide is from an entomopathogenic organism. In some embodiments, the entomopathogenic organism is selected from Bacillus thuringiensis, Pseudomonas aeruginosa, and Serratia marcescens. In a particular embodiment, the nucleic acid molecule encodes a Bacillus thuringiensis toxin.
In some embodiments, a recombinant microorganism is engineered to express a metabolic pathway which, when expressed, produces a small molecule that is toxic to an insect.
In exemplary embodiments, an insect pheromone produced by a recombinant microorganism described herein may be used to attract a pest insect, and subsequently, the pest insect is eradicated with a toxic substance, such as a toxic protein, peptide, or small molecule, which has been co-produced by a recombinant microorganism described herein.
Biosynthesis of Pheromones Using a Recombinant Microorganism
As discussed above, in a first aspect, the present disclosure relates to a recombinant microorganism capable of producing a mono- or poly-unsaturated C6-C24 fatty alcohol from an endogenous or exogenous source of saturated C6-C24 fatty acyl-CoA. An illustrative embodiment of the first aspect is shown in
Accordingly, in one embodiment, the recombinant microorganism expresses: (a) at least one exogenous nucleic acid molecule encoding a fatty acyl desaturase that catalyzes the conversion of a saturated C6-C24 fatty acyl-CoA to a corresponding mono- or poly-unsaturated C6-C24 fatty acyl-CoA; and (b) at least one exogenous nucleic acid molecule encoding a fatty alcohol forming fatty-acyl reductase that catalyzes the conversion of the mono- or poly-unsaturated C6-C24 fatty acyl-CoA from (a) into the corresponding mono- or poly-unsaturated C6-C24 fatty alcohol. In some embodiments, the saturated C6-C24 fatty acyl-CoA can be produced using endogenous enzymes in the host microorganism. In other embodiments, the saturated C6-C24 fatty acyl-CoA can be produced using one or more exogenous enzymes in the host microorganism.
As described above, a fatty acyl desaturase catalyzes the desaturation of the hydrocarbon chain on, e.g., a saturated fatty acyl-CoA molecule to generate a corresponding unsaturated fatty acyl CoA molecule. In some embodiments, an exogenous fatty acyl desaturase can be selected and expressed in a recombinant microorganism to catalyze the formation of at least one double bond in fatty acyl-CoA molecule having from 6 to 24 carbons in the hydrocarbon chain. Accordingly, in some embodiments, the fatty-acyl desaturase is a desaturase capable of utilizing a fatty acyl-CoA as a substrate that has a chain length of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbon atoms.
An exogenous fatty acyl desaturase described herein can be selected to catalyze the desaturation at a desired position on the hydrocarbon chain. Accordingly, in some embodiments, the fatty-acyl desaturase is capable of generating a double bond at position C5, C6, C7, C8, C9, C10, C11, C12, or C13, in the fatty acid or its derivatives, such as, for example, fatty acid CoA esters.
One or more than one fatty acyl-CoA desaturase can be expressed in the host to catalyze desaturation at multiple positions on the hydrocarbon chain. In some embodiments, the fatty acyl-CoA desaturase is heterologous to the host microorganism. Accordingly, various embodiments provide for recombinant microorganism comprised of at least one exogenous nucleic acid molecule, which encodes a fatty acyl desaturase that catalyzes the conversion of a saturated C6-C24 fatty acyl-CoA to a corresponding mono- or poly-unsaturated C6-C24 fatty acyl-CoA.
In one exemplary embodiment, the fatty-acyl desaturase is a Z11 desaturase. The Z11 fatty-acyl desaturase catalyze double bond formation between the 11th and 12th carbons in the substrate relative to the carbonyl group. In various embodiments described herein, the Z11 desaturase, or the nucleic acid sequence that encodes it, can be isolated from organisms of the species Agrotis segetum, Amyelois transitella, Argyrotaenia velutiana, Choristoneura rosaceana, Lampronia capitella, Trichoplusia ni, Helicoverpa zea, or Thalassiosira pseudonana. Further Z11-desaturases, or the nucleic acid sequences encoding them, can be isolated from Bombyx mori, Manduca sexta, Diatraea grandiosella, Earias insulana, Earias vittella, Plutella xylostellia, Bombyx mori or Diaphania nitidalis. In exemplary embodiments, the Z11 desaturase comprises a sequence selected from GenBank Accession Nos. JX679209, JX964774, AF416738, AF545481, EU152335, AAD03775, AAF81787, and AY493438. In some embodiments, a nucleic acid sequence encoding a Z11 desaturase from organisms of the species Agrotis segetum, Amyelois transitella, Argyrotaenia velutiana, Choristoneura rosaceana, Lampronia capitella, Trichoplusia ni, Helicoverpa zea, or Thalassiosira pseudonana is codon optimized. In some embodiments, the Z11 desaturase comprises a sequence selected from SEQ ID NOs: 9, 18, 24 and 26 from Trichoplusia ni. In some embodiments, the Z11 desaturase comprises an amino acid sequence set forth in SEQ ID NO: 49 from Trichoplusia ni. In other embodiments, the Z11 desaturase comprises a sequence selected from SEQ ID NOs: 10 and 16 from Agrotis segetum. In some embodiments, the Z11 desaturase comprises an amino acid sequence set forth in SEQ ID NO: 53 from Agrotis segetum. In some embodiments, the Z11 desaturase comprises a sequence selected from SEQ ID NOs: 11 and 23 from Thalassiosira pseudonana. In some embodiments, the Z11 desaturase comprises an amino acid sequence selected from SEQ ID NOs: 50 and 51 from Thalassiosira pseudonana. In certain embodiments, the Z11 desaturase comprises a sequence selected from SEQ ID NOs: 12, 17 and 30 from Amyelois transitella. In some embodiments, the Z11 desaturase comprises an amino acid sequence set forth in SEQ ID NO: 52 from Amyelois transitella. In further embodiments, the Z11 desaturase comprises a sequence selected from SEQ ID NOs: 13, 19, 25, 27 and 31 from Helicoverpa zea. In some embodiments, the Z11 desaturase comprises an amino acid sequence set forth in SEQ ID NO: 54 from Helicoverpa zea. In some embodiments, the Z11 desaturase comprises an amino acid sequence set forth in SEQ ID NO: 39 from S. inferens. In some embodiments, the Z11 desaturase comprises an amino acid sequence set forth in GenBank Accession nos. AF416738, AGH12217.1, A1121943.1, CAJ43430.2, AF441221, AAF81787.1, AF545481, AJ271414, AY362879, ABX71630.1, NP001299594.1, Q9N9Z8, ABX71630.1 and AIM40221.1. In some embodiments, the Z11 desaturase comprises a chimeric polypeptide. In some embodiments, a complete or partial Z11 desaturase is fused to another polypeptide. In certain embodiments, the N-terminal native leader sequence of a Z11 desaturase is replaced by an oleosin leader sequence from another species. In certain embodiments, the Z11 desaturase comprises a sequence selected from SEQ ID NOs: 15, 28 and 29. In some embodiments, the Z11 desaturase comprises an amino acid sequence selected from SEQ ID NOs: 61, 62, 63, 78, 79 and 80.
In certain embodiments, the Z11 desaturase catalyzes the conversion of a fatty acyl-CoA into a mono- or poly-unsaturated product selected from Z11-13:Acyl-CoA, E11-13:Acyl-CoA, (Z,Z)-7,11-13:Acyl-CoA, Z11-14:Acyl-CoA, E11-14:Acyl-CoA, (E,E)-9,11-14:Acyl-CoA, (E,Z)-9,11-14:Acyl-CoA, (Z,E)-9,11-14:Acyl-CoA, (Z,Z)-9,11-14:Acyl-CoA, (E,Z)-9,11-15:Acyl-CoA, (Z,Z)-9,11-15:Acyl-CoA, Z11-16:Acyl-CoA, E11-16:Acyl-CoA, (E,Z)-6,11-16:Acyl-CoA, (E,Z)-7,11-16:Acyl-CoA, (E,Z)-8,11-16:Acyl-CoA, (E,E)-9,11-16:Acyl-CoA, (E,Z)-9,11-16:Acyl-CoA, (Z,E)-9,11-16:Acyl-CoA, (Z,Z)-9,11-16:Acyl-CoA, (E,E)-11,13-16:Acyl-CoA, (E,Z)-11,13-16:Acyl-CoA, (Z,E)-11,13-16:Acyl-CoA, (Z,Z)-11,13-16:Acyl-CoA, (Z,E)-11,14-16:Acyl-CoA, (E,E,Z)-4,6,11-16:Acyl-CoA, (Z,Z,E)-7,11,13-16:Acyl-CoA, (E,E,Z,Z)-4,6,11,13-16:Acyl-CoA, Z11-17:Acyl-CoA, (Z,Z)-8,11-17:Acyl-CoA, Z11-18:Acyl-CoA, E11-18:Acyl-CoA, (Z,Z)-11,13-18:Acyl-CoA, (E,E)-11,14-18:Acyl-CoA, or combinations thereof.
In another exemplary embodiment, the fatty-acyl desaturase is a Z9 desaturase. The Z9 fatty-acyl desaturase catalyze double bond formation between the 9th and 10th carbons in the substrate relative to the carbonyl group. In various embodiments described herein, the Z9 desaturase, or the nucleic acid sequence that encodes it, can be isolated from organisms of the species Ostrinia furnacalis, Ostrinia nobilalis, Choristoneura rosaceana, Lampronia capitella, Helicoverpa assulta, or Helicoverpa zea. In exemplary embodiments, the Z9 desaturase comprises a sequence selected from GenBank Accession Nos. AY057862, AF243047, AF518017, EU152332, AF482906, and AAF81788. In some embodiments, a nucleic acid sequence encoding a Z9 desaturase is codon optimized. In some embodiments, the Z9 desaturase comprises a nucleotide sequence set forth in SEQ ID NO: 20 from Ostrinia furnacalis. In some embodiments, the Z9 desaturase comprises an amino acid sequence set forth in SEQ ID NO: 58 from Ostrinia furnacalis. In other embodiments, the Z9 desaturase comprises a nucleotide sequence set forth in SEQ ID NO: 21 from Lampronia capitella. In some embodiments, the Z9 desaturase comprises an amino acid sequence set forth in SEQ ID NO: 59 from Lampronia capitella. In some embodiments, the Z9 desaturase comprises a nucleotide sequence set forth in SEQ ID NO: 22 from Helicoverpa zea. In some embodiments, the Z9 desaturase comprises an amino acid sequence set forth in SEQ ID NO: 60 from Helicoverpa zea.
In certain embodiments, the Z9 desaturase catalyzes the conversion of a fatty acyl-CoA into a monounsaturated or polyunsaturated product selected from Z9-11:Acyl-CoA, Z9-12:Acyl-CoA, E9-12:Acyl-CoA, (E,E)-7,9-12:Acyl-CoA, (E,Z)-7,9-12:Acyl-CoA, (Z,E)-7,9-12:Acyl-CoA, (Z,Z)-7,9-12:Acyl-CoA, Z9-13:Acyl-CoA, E9-13:Acyl-CoA, (E,Z)-5,9-13:Acyl-CoA, (Z,E)-5,9-13:Acyl-CoA, (Z,Z)-5,9-13:Acyl-CoA, Z9-14:Acyl-CoA, E9-14:Acyl-CoA, (E,Z)-4,9-14:Acyl-CoA, (E,E)-9,11-14:Acyl-CoA, (E,Z)-9,11-14:Acyl-CoA, (Z,E)-9,11-14:Acyl-CoA, (Z,Z)-9,11-14:Acyl-CoA, (E,E)-9,12-14:Acyl-CoA, (Z,E)-9,12-14:Acyl-CoA, (Z,Z)-9,12-14:Acyl-CoA, Z9-15:Acyl-CoA, E9-15:Acyl-CoA, (Z,Z)-6,9-15:Acyl-CoA, Z9-16:Acyl-CoA, E9-16:Acyl-CoA, (E,E)-9,11-16:Acyl-CoA, (E,Z)-9,11-16:Acyl-CoA, (Z,E)-9,11-16:Acyl-CoA, (Z,Z)-9,11-16:Acyl-CoA, Z9-17:Acyl-CoA, E9-18:Acyl-CoA, Z9-18:Acyl-CoA, (E,E)-5,9-18:Acyl-CoA, (E,E)-9,12-18:Acyl-CoA, (Z,Z)-9,12-18:Acyl-CoA, (Z,Z, Z)-3,6,9-18:Acyl-CoA, (E,E,E)-9,12,15-18:Acyl-CoA, (Z,Z, Z)-9,12,15-18:Acyl-CoA, or combinations thereof.
Desaturation of a saturated C6-C24 fatty acyl-CoA can proceed through a plurality of reactions to produce a poly-unsaturated C6-C24 fatty acyl-CoA. In some embodiments, the recombinant microorganism may express a bifunctional desaturase capable of catalyzing the formation at least two double bonds. In some embodiments, the recombinant microorganism may express more than one exogenous nucleic acid molecule encoding more than one fatty-acyl desaturase that catalyzes the conversion of a saturated C6-C24 fatty acyl-CoA to a corresponding poly-unsaturated C6-C24 fatty acyl-CoA. For example, the recombinant microorganism may express an exogenous nucleic acid molecule encoding a Z11 desaturase and another exogenous nucleic acid molecule encoding a Z9 desaturase. Thus, the resultant poly-unsaturated fatty acyl-CoA would have a double bond between the 9th and 10th carbon and another double bond between the 11th and 12th carbon.
In some embodiments, the recombinant microorganism may express a fatty-acyl conjugase that acts independently or together with a fatty-acyl desaturase to catalyze the conversion of a saturated or monounsaturated fatty acyl-CoA to a conjugated polyunsaturated fatty acyl-CoA.
In one embodiment, the disclosure provides a recombinant microorganism capable of producing a polyunsaturated C6-C24 fatty alcohol from an endogenous or exogenous source of saturated or monounsaturated C6-C24 fatty acyl-CoA, wherein the recombinant microorganism expresses: (a) at least one exogenous nucleic acid molecule encoding a fatty acyl conjugase that catalyzes the conversion of a saturated or monounsaturated C6-C24 fatty acyl-CoA to a corresponding polyunsaturated C6-C24 fatty acyl-CoA; and (b) at least one exogenous nucleic acid molecule encoding a fatty alcohol forming fatty-acyl reductase that catalyzes the conversion of the polyunsaturated C6-C24 fatty acyl-CoA from (a) into the corresponding polyunsaturated C6-C24 fatty alcohol.
In another embodiment, the recombinant microorganism expresses at least two exogenous nucleic acid molecules encoding fatty-acyl conjugases that catalyze the conversion of a saturated or monounsaturated C6-C24 fatty acyl-CoA to a corresponding polyunsaturated C6-C24 fatty acyl-CoA.
In a further embodiment, the disclosure provides a recombinant microorganism capable of producing a polyunsaturated C6-C24 fatty alcohol from an endogenous or exogenous source of saturated or monounsaturated C6-C24 fatty acyl-CoA, wherein the recombinant microorganism expresses: (a) at least one exogenous nucleic acid molecule encoding a fatty-acyl desaturase and at least one exogenous nucleic acid molecule encoding a fatty acyl conjugase that catalyze the conversion of a saturated or monounsaturated C6-C24 fatty acyl-CoA to a corresponding polyunsaturated C6-C24 fatty acyl-CoA; and (b) at least one exogenous nucleic acid molecule encoding a fatty alcohol forming fatty-acyl reductase that catalyzes the conversion of the polyunsaturated C6-C24 fatty acyl-CoA from (a) into the corresponding polyunsaturated C6-C24 fatty alcohol.
In another embodiment, the recombinant microorganism expresses at least two exogenous nucleic acid molecules encoding fatty-acyl desaturases and at least two exogenous nucleic acid molecules encoding fatty-acyl conjugases that catalyze the conversion of a saturated or monounsaturated C6-C24 fatty acyl-CoA to a corresponding polyunsaturated C6-C24 fatty acyl-CoA.
In yet a further embodiment, the fatty-acyl conjugase is a conjugase capable of utilizing a fatty acyl-CoA as a substrate that has a chain length of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbon atoms.
In certain embodiments, the conjugase, or the nucleic acid sequence that encodes it, can be isolated from organisms of the species Cydia pomonella, Cydia nigricana, Lobesia botrana, Myelois cribrella, Plodia interpunctella, Dendrolimus punctatus, Lampronia capitella, Spodoptera litura, Amyelois transitella, Manduca sexta, Bombyx mori, Calendula officinalis, Trichosanthes kirilowii, Punica granatum, Momordica charantia, Impatiens balsamina, and Epiphyas postvittana. In exemplary embodiments, the conjugase comprises a sequence selected from GenBank Accession No. or Uniprot database: A0A059TBF5, A0A0M3L9E8, A0A0M3L9S4, A0A0M3LAH8, A0A0M3LAS8, A0A0M3LAH8, B6CBS4, XP_013183656.1, XP_004923568.2, ALA65425.1, NP_001296494.1, NP_001274330.1, Q4A181, Q75PL7, Q9FPP8, AY178444, AY178446, AF182521, AF182520, Q95UJ3.
As described above, a fatty acyl reductase catalyzes the reduction of a carbonyl group, e.g., on an unsaturated fatty acyl-CoA molecule to generate a corresponding unsaturated fatty acid molecule. In some embodiments, the fatty alcohol forming fatty acyl CoA reductase is heterologous to the microorganism. Accordingly, various embodiments provide for recombinant microorganism comprised of at least one exogenous nucleic acid molecule, which encodes a fatty alcohol forming fatty acyl reductase that catalyzes the reduction of a carbonyl group on an unsaturated fatty acyl-CoA molecule to generate a corresponding unsaturated fatty acid molecule.
In some embodiments, the fatty acyl reductase is from an organism of the species Agrotis segetum, Spodoptera exigua, Spodoptera littoralis, Euglena gracilis, Yponomeuta evonymellus and Helicoverpa armigera. In some embodiments, a nucleic acid sequence encoding a fatty-acyl reductase is codon optimized. In some embodiments, the fatty acyl reductase comprises a sequence set forth in SEQ ID NO: 1 from Agrotis segetum. In some embodiments, the fatty acyl reductase comprises an amino acid sequence set forth in SEQ ID NO: 55 from Agrotis segetum. In other embodiments, the fatty acyl reductase comprises a sequence set forth in SEQ ID NO: 2 from Spodoptera littoralis. In other embodiments, the fatty acyl reductase comprises an amino acid sequence set forth in SEQ ID NO: 56 from Spodoptera littoralis. In some embodiments, the fatty acyl reductase comprises a sequence selected from SEQ ID NOs: SEQ ID NOs: 3, 32, 40, 72, 74, 76 and 81. In some embodiments, the fatty acyl reductase comprises an amino acid sequence set forth in SEQ ID NO: 55 from Agrotis segetum. In other embodiments, the fatty acyl reductase comprises an amino acid sequence set forth in SEQ ID NO: 56 from Spodoptera littoralis. In some embodiments, the fatty acyl reductase comprises an amino acid sequence selected from SEQ ID NOs: 41 and 57 from Helicoverpa armigera. In some embodiments, the fatty acyl reductase comprises an amino acid sequence selected from SEQ ID NOs: 73 and 82 from Spodoptera exigua. In some embodiments, the fatty acyl reductase comprises an amino acid sequence set forth in SEQ ID NO: 75 from Euglena gracilis. In some embodiments, the fatty acyl reductase comprises an amino acid sequence set forth in SEQ ID NO: 77 from Yponomeuta evonymellus.
In some embodiments, the production of unsaturated fatty alcohols in a recombinant microorganism comprises the expression of one or more mutant FARs. In certain embodiments, Helicoverpa amigera fatty acyl-CoA reductase (HaFAR) variants are provided which show a net increase in fatty alcohol produced compared to the wild type Helicoverpa amigera fatty acyl-CoA reductase encoded by an amino acid sequence set forth in SEQ ID NO: 41. In some embodiments, the increased enzymatic activity is a net activity increase in amount of fatty alcohol produced relative to the amount of fatty alcohol produced by a wild type enzymatic activity of HaFAR encoded by an amino acid sequence set forth in SEQ ID NO: 41. In some embodiments, a wild type HaFAR comprises a nucleotide sequence set forth in SEQ ID NO: 90. In some embodiments, a variant of a wild type HaFAR encoded by an amino acid sequence set forth in SEQ ID NO: 41 comprises point mutations at the following positions: S60X, S195X, S298X, S378X, S394X, S418X, and S453X, wherein X comprises the amino acids F, L, M, I, V, P, T, A, Y, K, H, N, Q, K, D, E, C, W, R. In some embodiments, a variant of a wild type HaFAR encoded by an amino acid sequence set forth in SEQ ID NO: 41 comprises a combination of point mutations selected from mutations at the following amino acid positions: S60X, S195X, S298X, S378X, S394X, S418X, and S453X, wherein X comprises the amino acids F, L, M, I, V, P, T, A, Y, K, H, N, Q, K, D, E, C, W, R. In some embodiments, the fatty acyl reductase is a mutated fatty acyl reductase and comprises an amino acid sequence selected from SEQ ID NOs: 42-48. In some embodiments, the fatty acyl reductase is a mutated fatty acyl reductase and comprises nucleotide sequence selected from SEQ ID NOs: 83-89.
In exemplary embodiments, the fatty-acyl reductase catalyzes the conversion of a mono- or poly-unsaturated fatty acyl-CoA into a fatty alcohol product selected from (Z)-3-hexenol, (Z)-3-nonenol, (Z)-5-decenol, (E)-5-decenol, (Z)-7-dodecenol, (E)-8-dodecenol, (Z)-8-dodecenol, (Z)-9-dodecenol, (Z)-9-tetradecenol, (Z)-9-hexadecenol, (Z)-11-tetradecenol, (Z)-7-hexadecenol, (Z)-11-hexadecenol, (E)-11-tetradecenol, or (Z,Z)-11,13-hexadecadienol, (11Z,13E)-hexadecadienol, (E,E)-8,10-dodecadienol, (E,Z)-7,9-dodecadienol, (Z)-13-octadecenol, or combinations thereof.
In some embodiments, a recombinant microorganism described herein can include a plurality of fatty acyl reductases. Accordingly, in such embodiments, the recombinant microorganism expresses at least two exogenous nucleic acid molecules, which encode fatty-acyl reductases that catalyze the conversion of the mono- or poly-unsaturated C6-C24 fatty acyl-CoA into the corresponding mono- or poly-unsaturated C6-C24 fatty alcohol.
In a further embodiment, the disclosure provides a recombinant microorganism capable of producing a mono- or poly-unsaturated ≤C18 fatty alcohol from an endogenous or exogenous source of saturated C6-C24 fatty acid, wherein the recombinant microorganism comprises: (a) at least one exogenous nucleic acid molecule encoding a fatty acyl desaturase that catalyzes the conversion of a saturated C6-C24 fatty acyl-CoA to a corresponding mono- or poly-unsaturated C6-C24 fatty acyl-CoA; (b) at least one exogenous nucleic acid molecule encoding an acyl-CoA oxidase that catalyzes the conversion of the mono- or poly-unsaturated C6-C24 fatty acyl-CoA from (a) into a mono- or poly-unsaturated ≤C18 fatty acyl-CoA after one or more successive cycle of acyl-CoA oxidase activity, with a given cycle producing a mono- or poly-unsaturated C4-C22 fatty acyl-CoA intermediate with a two carbon truncation relative to a starting mono- or poly-unsaturated C6-C24 fatty acyl-CoA substrate in that cycle; and (c) at least one exogenous nucleic acid molecule encoding a fatty alcohol forming fatty acyl reductase that catalyzes the conversion of the mono- or poly-unsaturated ≤C18 fatty acyl-CoA from (b) into the corresponding mono- or poly-unsaturated ≤C18 fatty alcohol. In some embodiments, the fatty acyl desaturase is selected from an Argyrotaenia velutinana, Spodoptera litura, Sesamia inferens, Manduca sexta, Ostrinia nubilalis, Helicoverpa zea, Choristoneura rosaceana, Drosophila melanogaster, Spodoptera littoralis, Lampronia capitella, Amyelois transitella, Trichoplusia ni, Agrotis segetum, Ostrinia furnicalis, and Thalassiosira pseudonana derived fatty acyl desaturase. In some embodiments, the fatty acyl desaturase has 95% sequence identity to a fatty acyl desaturase selected from the group consisting of: SEQ ID NOs: 39, 49-54, 58-63, 78-80 and GenBank Accession nos. AF416738, AGH12217.1, AI121943.1, CAJ43430.2, AF441221, AAF81787.1, AF545481, AJ271414, AY362879, ABX71630.1, NP001299594.1, Q9N9Z8, ABX71630.1 and AIM40221.1. In some embodiments, the acyl-CoA oxidase is selected from Table 5a. In other embodiments, the fatty alcohol forming fatty acyl reductase is selected from an Agrotis segetum, Spodoptera exigua, Spodoptera littoralis, Euglena gracilis, Yponomeuta evonymellus and Helicoverpa armigera derived fatty alcohol forming fatty acyl reductase. In further embodiments, the fatty alcohol forming fatty acyl reductase has 95% sequence identity to a fatty alcohol forming fatty acyl reductase selected from the group consisting of: SEQ ID NOs: 1-3, 32, 41-48, 55-57, 73, 75, 77 and 82. In some embodiments, the recombinant microorganism is a yeast selected from the group consisting of Yarrowia lipolytica, Saccharomyces cerevisiae, Candida albicans, Candida tropicalis and Candida viswanathii.
In some embodiments, the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an acyltransferase that preferably stores ≤C18 fatty acyl-CoA. In some embodiments, the acyltransferase is selected from the group consisting of glycerol-3-phosphate acyl transferase (GPAT), lysophosphatidic acid acyltransferase (LPAAT), glycerolphospholipid acyltransferase (GPLAT) and diacylglycerol acyltransferases (DGAT). In some preferred embodiments, the acyltransferase is selected from Table 5b.
In some embodiments, the coexpression of a wax esterase would allow the storage of fatty alcohols and fatty acids in a 1:1 ratio. In combination with TAG storage that could lead to interesting ratios of TAG and fatty alcohols which could subsequently be used for different product streams. Examples for waxester synthases: Homo sapiens AWAT2 (XM_011530876.2), Mus musculus (AAT68766.1) Euglena gracilis WS (ADI60058.1), Euglena gracilis WSD2 (BAV82975.1), Euglena gracilis WSDS (BAV82978.1).
In some embodiments, the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an acylglycerol lipase that preferably hydrolyzes ester bonds of >C16, of >C14, of >C12 or of >C10 acylglycerol substrates. In some embodiments, the acylglycerol lipase is selected from Table 5c.
In some embodiments, the recombinant microorganism comprises a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous enzymes that catalyzes a reaction in a pathway that competes with the biosynthesis pathway for the production of a mono- or poly-unsaturated ≤C18 fatty alcohol. In further embodiments, the recombinant microorganism comprises a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous enzyme selected from: (i) one or more acyl-CoA oxidase; (ii) one or more acyltransferase; (iii) one or more acylglycerol lipase and/or sterol ester esterase; (iv) one or more (fatty) alcohol dehydrogenase; (v) one or more (fatty) alcohol oxidase; and (vi) one or more cytochrome P450 monooxygenase.
In some preferred embodiments, one or more genes of the microbial host encoding acyl-CoA oxidases are deleted or down-regulated to eliminate or reduce the truncation of desired fatty acyl-CoAs beyond a desired chain-length. In some embodiments, the recombinant microorganism comprises a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous acyl-CoA oxidase enzyme selected from the group consisting of Y. lipolytica POX1 (YALI0E32835g), Y. lipolytica POX2 (YALI0F10857g), Y. lipolytica POX3 (YALI0D24750g), Y. lipolytica POX4 (YALI0E27654g), Y. lipolytica POX5 (YALI0C23859g), Y. lipolytica POX6 (YALI0E06567g); S. cerevisiae POX1 (YGL205W); Candida POX2 (CaO19.1655, CaO19.9224, CTRG_02374, M18259), Candida POX4 (CaO19.1652, CaO19.9221, CTRG_02377, M12160), and Candida POX5 (CaO19.5723, CaO19.13146, CTRG_02721, M12161).
In some embodiments, a recombinant microorganism capable of producing a mono- or poly-unsaturated C6-C24 fatty alcohol, fatty aldehyde and/or fatty acetate from an endogenous or exogenous source of saturated C6-C24 fatty acid is provided, wherein the recombinant microorganism expresses one or more acyl-CoA oxidase enzymes, and wherein the recombinant microorganism is manipulated to delete, disrupt, mutate, and/or reduce the activity of one or more endogenous acyl-CoA oxidase enzymes. In some embodiments, the one or more acyl-CoA oxidase enzymes being expressed are different from the one or more endogenous acyl-CoA oxidase enzymes being deleted or downregulated. In other embodiments, the one or more acyl-CoA oxidase enzymes that are expressed regulate chain length of the mono- or poly-unsaturated C6-C24 fatty alcohol, fatty aldehyde and/or fatty acetate. In other embodiments, the one or more acyl-CoA oxidase enzymes being expressed are selected from Table 5a.
In some embodiments, the recombinant microorganism comprises a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous acyltransferase enzyme selected from the group consisting of Y. lipolytica YALI0C00209g, Y. lipolytica YALI0E18964g, Y. lipolytica YALI0F19514g, Y. lipolytica YALI0C14014g, Y. lipolytica YALI0E16797g, Y. lipolytica YALI0E32769g, and Y. lipolytica YALI0D07986g, S. cerevisiae YBL011w, S. cerevisiae YDL052c, S. cerevisiae YOR175C, S. cerevisiae YPR139C, S. cerevisiae YNR008w, and S. cerevisiae YOR245c, and Candida 1503_02577, Candida CTRG_02630, Candida CaO19.250, Candida CaO19.7881, Candida CTRG_02437, Candida CaO19.1881, Candida CaO19.9437, Candida CTRG_01687, Candida CaO19.1043, Candida CaO19.8645, Candida CTRG_04750, Candida CaO19.13439, Candida CTRG_04390, Candida CaO19.6941, Candida CaO19.14203, and Candida CTRG_06209.
In some embodiments, a recombinant microorganism capable of producing a mono- or poly-unsaturated ≤C18 fatty alcohol, fatty aldehyde and/or fatty acetate from an endogenous or exogenous source of saturated C6-C24 fatty acid is provided, wherein the recombinant microorganism expresses one or more acyltransferase enzymes, and wherein the recombinant microorganism is manipulated to delete, disrupt, mutate, and/or reduce the activity of one or more endogenous acyltransferase enzymes. In some preferred embodiments, one or more genes of the microbial host encoding GPATs, LPAATs, GPLATs and/or DGATs are deleted or downregulated, and replaced with one or more GPATs, LPAATs, GPLATs, or DGATs which prefer to store short-chain fatty acyl-CoAs. In some embodiments, the one or more acyltransferase enzymes being expressed are different from the one or more endogenous acyltransferase enzymes being deleted or downregulated. In other embodiments, the one or more acyltransferase enzymes being expressed are selected from Table 5b.
In some preferred embodiments, one or more genes of the microbial host encoding acylglycerol lipases (mono-, di-, or triacylglycerol lipases) and sterol ester esterases are deleted or downregulated and replaced with one or more acylglycerol lipases which prefer long chain acylglycerol substrates. In some embodiments, the recombinant microorganism comprises a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous acylglycerol lipase and/or sterol ester esterase enzyme selected from the group consisting of Y. lipolytica YALI0E32035g, Y. lipolytica YALI0D17534g, Y. lipolytica YALI0F10010g, Y. lipolytica YALI0C14520g, and Y. lipolytica YALI0E00528g, S. cerevisiae YKL140w, S. cerevisiae YMR313c, S. cerevisiae YKR089c, S. cerevisiae YOR081c, S. cerevisiae YKL094W, S. cerevisiae YLL012W, and S. cerevisiae YLR020C, and Candida CaO19.2050, Candida CaO19.9598, Candida CTRG_01138, Candida W5Q_03398, Candida CTRG_00057, Candida CaO19.5426, Candida CaO19.12881, Candida CTRG_06185, Candida CaO19.4864, Candida CaO19.12328, Candida CTRG_03360, Candida CaO19.6501, Candida CaO19.13854, Candida CTRG_05049, Candida CaO19.1887, Candida CaO19.9443, Candida CTRG_01683, and Candida CTRG_04630.
In some embodiments, the recombinant microorganism comprises a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous cytochrome P450 monooxygenases selected from the group consisting of Y. lipolytica YALI0E25982g (ALK1), Y. lipolytica YALI0F01320g (ALK2), Y. lipolytica YALI0E23474g (ALK3), Y. lipolytica YALI0B13816g (ALK4), Y. lipolytica YALI0B13838g (ALK5), Y. lipolytica YALI0B01848g (ALK6), Y. lipolytica YALI0A15488g (ALK7), Y. lipolytica YALI0C12122g (ALK8), Y. lipolytica YALI0B06248g (ALK9), Y. lipolytica YALI0B20702g (ALK10), Y. lipolytica YALI0C10054g (ALK11) and Y. lipolytica YALI0A20130g (ALK12).
In some embodiments, a recombinant microorganism capable of producing a mono- or poly-unsaturated ≤C18 fatty alcohol, fatty aldehyde and/or fatty acetate from an endogenous or exogenous source of saturated C6-C24 fatty acid is provided, wherein the recombinant microorganism expresses one or more acylglycerol lipase and/or sterol ester esterase enzymes, and wherein the recombinant microorganism is manipulated to delete, disrupt, mutate, and/or reduce the activity of one or more endogenous acylglycerol lipase and/or sterol ester esterase enzymes. In some embodiments, the one or more acylglycerol lipase and/or sterol ester esterase enzymes being expressed are different from the one or more endogenous acylglycerol lipase and/or sterol ester esterase enzymes being deleted or downregulated. In some embodiments, the one or more endogenous or exogenous acylglycerol lipase and/or sterol ester esterase enzymes being expressed prefer to hydrolyze ester bonds of long-chain acylglycerols. In other embodiments, the one or more acylglycerol lipase and/or sterol ester esterase enzymes being expressed are selected from Table 5c.
In some embodiments, the fatty acyl desaturase catalyzes the conversion of a fatty acyl-CoA into a mono- or poly-unsaturated intermediate selected from E5-10:Acyl-CoA, E7-12:Acyl-CoA, E9-14:Acyl-CoA, E11-16:Acyl-CoA, E13-18:Acyl-CoA, Z7-12:Acyl-CoA, Z9-14:Acyl-CoA, Z11-16:Acyl-CoA, Z13-18:Acyl-CoA, Z8-12:Acyl-CoA, Z10-14:Acyl-CoA, Z12-16:Acyl-CoA, Z14-18:Acyl-CoA, Z7-10:Acyl-coA, Z9-12:Acyl-CoA, Z11-14:Acyl-CoA, Z13-16:Acyl-CoA, Z15-18:Acyl-CoA, E7-10:Acyl-CoA, E9-12:Acyl-CoA, E11-14:Acyl-CoA, E13-16:Acyl-CoA, E15-18:Acyl-CoA, E5Z7-12:Acyl-CoA, E7Z9-12:Acyl-CoA, E9Z11-14:Acyl-CoA, E11Z13-16:Acyl-CoA, E13Z15-18:Acyl-CoA, E6E8-10:Acyl-CoA, E8E10-12:Acyl-CoA, E10E12-14:Acyl-CoA, E12E14-16:Acyl-CoA, Z5E8-10:Acyl-CoA, Z7E10-12:Acyl-CoA, Z9E12-14:Acyl-CoA, Z11E14-16:Acyl-CoA, Z13E16-18:Acyl-CoA, Z3-10:Acyl-CoA, Z5-12:Acyl-CoA, Z7-14:Acyl-CoA, Z9-16:Acyl-CoA, Z11-18:Acyl-CoA, Z3Z5-10:Acyl-CoA, Z5Z7-12:Acyl-CoA, Z7Z9-14:Acyl-CoA, Z9Z11-16:Acyl-CoA, Z11Z13-16:Acyl-CoA, and Z13Z15-18:Acyl-CoA. In further embodiments, the mono- or poly-unsaturated ≤C18 fatty alcohol is selected from the group consisting of E5-10:OH, Z8-12:OH, Z9-12:OH, Z11-14:OH, Z11-16:OH, E11-14:OH, E8E10-12:OH, E7Z9-12:OH, Z11Z13-16OH, Z9-14:OH, Z9-16:OH, and Z13-18:OH.
In some embodiments, the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an aldehyde forming fatty acyl-CoA reductase capable of catalyzing the conversion of the mono- or poly-unsaturated ≤C18 fatty alcohol into a corresponding ≤C18 fatty aldehyde. In some preferred embodiments, the aldehyde forming fatty acyl-CoA reductase is selected from the group consisting of Acinetobacter calcoaceticus A0A1C4HN78, A. calcoaceticus N9DA85, A. calcoaceticus R8XW24, A. calcoaceticus A0A1A0GGM5, A. calcoaceticus A0A117N158, and Nostoc punctiforme YP_001865324. In some embodiments, the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an alcohol oxidase or an alcohol dehydrogenase capable of catalyzing the conversion of the mono- or poly-unsaturated ≤C18 fatty alcohol into a corresponding ≤C18 fatty aldehyde. In some preferred embodiments, the ≤C18 fatty aldehyde is selected from the group consisting of Z9-16:Ald, Z11-16:Ald, Z11Z13-16:Ald, and Z13-18:Ald.
In some embodiments, the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an acetyl transferase capable of catalyzing the conversion of the mono- or poly-unsaturated ≤C18 fatty alcohol into a corresponding ≤C18 fatty acetate. In some embodiments, the acetyl transferase is selected from Table 5d. In some preferred embodiments, the ≤C18 fatty acetate is selected from the group consisting of E5-10:Ac, Z7-12:Ac, Z8-12:Ac, Z9-12:Ac, E7Z9-12:Ac, Z9-14:Ac, Z9E12-14:Ac, Z11-14:Ac, E11-14:Ac, Z9-16:Ac, and Z11-16:Ac.
In some embodiments, the recombinant microorganism further comprises: at least one endogenous or exogenous nucleic acid molecule encoding an enzyme selected from an alcohol oxidase, an alcohol dehydrogenase, and an aldehyde forming fatty acyl-CoA reductase capable of catalyzing the conversion of the mono- or poly-unsaturated ≤C18 fatty alcohol into a corresponding ≤C18 fatty aldehyde; and at least one endogenous or exogenous nucleic acid molecule encoding an acetyl transferase capable of catalyzing the conversion of the mono- or poly-unsaturated ≤C18 fatty alcohol into a corresponding ≤C18 fatty acetate. In some preferred embodiments, the mono- or poly-unsaturated ≤C18 fatty aldehyde and ≤C18 fatty acetate is selected from the group consisting of E5-10:Ac, Z7-12:Ac, Z8-12:Ac, Z9-12:Ac, E7Z9-12:Ac, Z9-14:Ac, Z9E12-14:Ac, E11-14:Ac, Z11-14:Ac, Z11-16:Ac, Z9-16:Ac, Z9-16:Ald, Z11-16:Ald, Z11Z13-16:Ald, and Z13-18:Ald.
In a further embodiment, the disclosure provides a recombinant Yarrowia lipolytica microorganism capable of producing a mono- or poly-unsaturated C6-C24 fatty alcohol from an endogenous or exogenous source of saturated C6-C24 fatty acid, wherein the recombinant Yarrowia lipolytica microorganism comprises: (a) at least one nucleic acid molecule encoding a fatty acyl desaturase having 95% sequence identity to a fatty acyl desaturase selected from the group consisting of SEQ ID NOs: 54, 60, 62, 78, 79, 80, 95, 97, 99, 101, 103, and 105 that catalyzes the conversion of a saturated C6-C24 fatty acyl-CoA to a corresponding mono- or poly-unsaturated C6-C24 fatty acyl-CoA; and (b) at least one nucleic acid molecule encoding a fatty alcohol forming fatty acyl reductase having 95% sequence identity to a fatty alcohol forming fatty acyl reductase selected from the group consisting of SEQ ID NOs: 41-48, 57, 73, 75 and 77 that catalyzes the conversion of the mono- or poly-unsaturated C6-C24 fatty acyl-CoA from (a) into the corresponding mono- or poly-unsaturated C6-C24 fatty alcohol.
In some embodiments, the recombinant Yarrowia lipolytica microorganism comprises a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous enzymes that catalyzes a reaction in a pathway that competes with the biosynthesis pathway for the production of a mono- or poly-unsaturated C6-C24 fatty alcohol. In some preferred embodiments, the recombinant Yarrowia lipolytica microorganism comprises a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous enzyme selected from the following: (i) one or more acyl-CoA oxidase selected from the group consisting of YALI0E32835g (POX1), YALI0F10857g (POX2), YALI0D24750g (POX3), YALI0E27654g (POX4), YALI0C23859g (POX5), YALI0E06567g (POX6); (ii) one or more (fatty) alcohol dehydrogenase selected from the group consisting of YALI0F09603g (FADH), YALI0D25630g (ADH1), YALI0E17787g (ADH2), YALI0A16379g (ADH3), YALI0E15818g (ADH4), YALI0D02167g (ADH5), YALI0A15147g (ADH6), YALI0E07766g (ADH7); (iii) a (fatty) alcohol oxidase YALI0B14014g (FAO1); (iv) one or more cytochrome P450 enzyme selected from the group consisting of YALI0E25982g (ALK1), YALI0F01320g (ALK2), YALI0E23474g (ALK3), YALI0B13816g (ALK4), YALI0B13838g (ALK5), YALI0B01848g (ALK6), YALI0A15488g (ALK7), (YALI0C12122g (ALK8), YALI0B06248g (ALK9), YALI0B20702g (ALK10), YALI0C10054g (ALK11) and YALI0A20130g (Alk12); and (v) one or more diacylglycerol acyltransferase selected from the group consisting of YALI0E32791g (DGA1) and YALI0D07986g (DGA2). In other preferred embodiments, the recombinant Yarrowia lipolytica microorganism comprises a deletion of one or more endogenous enzyme selected from the following: (i) one or more acyl-CoA oxidase selected from the group consisting of YALI0E32835g (POX1), YALI0F10857g (POX2), YALI0D24750g (POX3), YALI0E27654g (POX4), YALI0C23859g (POX5), YALI0E06567g (POX6); (ii) one or more (fatty) alcohol dehydrogenase selected from the group consisting of YALI0F09603g (FADH), YALI0D25630g (ADH1), YALI0E17787g (ADH2), YALI0A16379g (ADH3), YALI0E15818g (ADH4), YALI0D02167g (ADH5), YALI0A15147g (ADH6), YALI0E07766g (ADH7); (iii) a (fatty) alcohol oxidase YALI0B14014g (FAO1); (iv) one or more cytochrome P450 enzyme selected from the group consisting of YALI0E25982g (ALK1), YALI0F01320g (ALK2), YALI0E23474g (ALK3), YALI0B13816g (ALK4), YALI0B13838g (ALK5), YALI0B01848g (ALK6), YALI0A15488g (ALK7), (YALI0C12122g (ALK8), YALI0B06248g (ALK9), YALI0B20702g (ALK10), YALI0C10054g (ALK11) and YALI0A20130g (Alk12); and (v) one or more diacylglycerol acyltransferase selected from the group consisting of YALI0E32791g (DGA1) and YALI0D07986g (DGA2).
In some embodiments, the fatty acyl desaturase catalyzes the conversion of a saturated fatty acyl-CoA into a mono- or poly-unsaturated intermediate selected from Z9-14:Acyl-CoA, Z11-14:Acyl-CoA, E11-14:Acyl-CoA, Z9-16:Acyl-CoA, and Z11-16:Acyl-CoA. In other embodiments, the mono- or poly-unsaturated C6-C24 fatty alcohol is selected from the group consisting of Z9-14:OH, Z11-14:OH, E11-14:OH, Z9-16:OH, Z11-16:OH, Z11Z13-16:OH, and Z13-18:OH.
In some embodiments, the recombinant Yarrowia lipolytica microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an alcohol oxidase or an alcohol dehydrogenase capable of catalyzing the conversion of the mono- or poly-unsaturated C6-C24 fatty alcohol into a corresponding C6-C24 fatty aldehyde. In some embodiments, the alcohol dehydrogenase is selected from Table 3a. In some embodiments, the C6-C24 fatty aldehyde is selected from the group consisting of Z9-14:Ald, Z11-14:Ald, E11-14:Ald, Z9-16:Ald, Z11-16:Ald, Z11Z13-16:Ald and Z13-18:Ald.
In some embodiments, the recombinant Yarrowia lipolytica microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an acetyl transferase capable of catalyzing the conversion of the mono- or poly-unsaturated C6-C24 fatty alcohol into a corresponding C6-C24 fatty acetate. In some embodiments, the acetyl transferase is selected from Table 5d. In some embodiments, the C6-C24 fatty acetate is selected from the group consisting of Z9-14:Ac, Z11-14:Ac, E11-14:Ac, Z9-16:Ac, Z11-16:Ac, Z11Z13-16:Ac, and Z13-18:Ac.
In some embodiments, the recombinant Yarrowia lipolytica microorganism further comprises: at least one endogenous or exogenous nucleic acid molecule encoding an alcohol oxidase or an alcohol dehydrogenase capable of catalyzing the conversion of the mono- or poly-unsaturated C6-C24 fatty alcohol into a corresponding C6-C24 fatty aldehyde; and at least one endogenous or exogenous nucleic acid molecule encoding an acetyl transferase capable of catalyzing the conversion of the mono- or poly-unsaturated C6-C24 fatty alcohol into a corresponding C6-C24 fatty acetate. In some embodiments, the mono- or poly-unsaturated C6-C24 fatty aldehyde and C6-C24 fatty acetate is selected from the group consisting of Z9-14:Ac, Z11-14:Ac, E11-14:Ac, Z9-16:Ac, Z11-16:Ac, Z11Z13-16:Ac, Z13-18:Ac, Z9-14:Ald, Z11-14:Ald, E11-14:Ald, Z9-16:Ald, Z11-16:Ald, Z11Z13-16:Ald and Z13-18:Ald.
In some embodiments, the fatty acyl desaturase does not comprise a fatty acyl desaturase comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 64, 65, 66 and 67. In other embodiments, the fatty acyl desaturase does not comprise a fatty acyl desaturase selected from an Amyelois transitella, Spodoptera littoralis, Agrotis segetum, or Trichoplusia ni derived desaturase.
In some embodiments, the disclosure provides a method of engineering a Yarrowia lipolytica microorganism that is capable of producing a mono- or poly-unsaturated C6-C24 fatty alcohol from an endogenous or exogenous source of saturated C6-C24 fatty acid, wherein the method comprises introducing into the Yarrowia lipolytica microorganism the following: (a) at least one nucleic acid molecule encoding a fatty acyl desaturase having 95% sequence identity to a fatty acyl desaturase selected from the group consisting of SEQ ID NOs: 39, 54, 60, 62, 78, 79, 80, 95, 97, 99, 101, 103, and 105 that catalyzes the conversion of a saturated C6-C24 fatty acyl-CoA to a corresponding mono- or poly-unsaturated C6-C24 fatty acyl-CoA; and (b) at least one nucleic acid molecule encoding a fatty alcohol forming fatty acyl reductase having 95% sequence identity to a fatty alcohol forming fatty acyl reductase selected from the group consisting of SEQ ID NOs: 41-48, 57, 73, 75 and 77 that catalyzes the conversion of the mono- or poly-unsaturated C6-C24 fatty acyl-CoA from (a) into the corresponding mono- or poly-unsaturated C6-C24 fatty alcohol. In some embodiments, the microorganism is MATA ura3-302::SUC2 Δpox1 Δpox2 Δpox3 Δpox4 Δpox5 Δpox6 Δfadh Δadh1 Δadh2 Δadh3 Δadh4 Δadh5 Δadh6 Δadh7 Δfao1::URA3.
In some embodiments, the disclosure provides a method of producing a mono- or poly-unsaturated C6-C24 fatty alcohol, fatty aldehyde or fatty acetate from an endogenous or exogenous source of saturated C6-C24 fatty acid, comprising: cultivating a recombinant microorganism described herein in a culture medium containing a feedstock that provides a carbon source adequate for the production of the mono- or poly-unsaturated C6-C24 fatty alcohol, fatty aldehyde or fatty acetate. In some embodiments, the method further comprises a step of recovering the mono- or poly-unsaturated C6-C24 fatty alcohol, fatty aldehyde or fatty acetate. In further embodiments, the recovery step comprises distillation. In yet further embodiments, the recovery step comprises membrane-based separation.
In some embodiments, the mono- or poly-unsaturated C6-C24 fatty alcohol is converted into a corresponding C6-C24 fatty aldehyde using chemical methods. In further embodiments, the chemical methods are selected from TEMPO-bleach, TEMPO-copper-air, TEMPO-PhI(OAc)2, Swern oxidation and noble metal-air. In some embodiments, the mono- or poly-unsaturated C6-C24 fatty alcohol is converted into a corresponding C6-C24 fatty acetate using chemical methods. In further embodiments, the chemical methods utilize a chemical agent selected from the group consisting of acetyl chloride, acetic anhydride, butyryl chloride, butyric anhydride, propanoyl chloride and propionic anhydride in the presence of 4-N,N-dimethylaminopyridine (DMAP) or sodium acetate to esterify the mono- or poly-unsaturated C6-C24 fatty alcohol to the corresponding C6-C24 fatty acetate.
As discussed above, in a second aspect, the application relates to a recombinant microorganism capable of producing an unsaturated C6-C24 fatty alcohol from an endogenous or exogenous source of C6-C24 fatty acid. An illustrative embodiment of the second aspect is shown in
In one embodiment, the recombinant microorganism expresses (a): at least one exogenous nucleic acid molecule encoding an acyl-ACP synthetase that catalyzes the conversion of a C6-C24 fatty acid to a corresponding saturated C6-C24 fatty acyl-ACP; (b) at least one exogenous nucleic acid molecule encoding a fatty-acyl-ACP desaturase that catalyzes the conversion of a saturated C6-C24 fatty acyl-ACP to a corresponding mono- or poly-unsaturated C6-C24 fatty acyl-ACP; (c) one or more endogenous or exogenous nucleic acid molecules encoding a fatty acid synthase complex that catalyzes the conversion of the mono- or poly-unsaturated C6-C24 fatty acyl-ACP from (b) to a corresponding mono- or poly-unsaturated C6-C24 fatty acyl-ACP with a two carbon elongation relative to the product of (b); (d): at least one exogenous nucleic acid molecule encoding a fatty aldehyde forming fatty-acyl reductase that catalyzes the conversion of the mono- or poly-unsaturated C6-C24 fatty acyl-ACP from (c) into a corresponding mono- or poly-unsaturated C6-C24 fatty aldehyde; and (e) at least one endogenous or exogenous nucleic acid molecule encoding a dehydrogenase that catalyzes the conversion of the mono- or poly-unsaturated C6-C24 fatty aldehyde C6-C24 from (d) into a corresponding mono- or poly-unsaturated C6-C24 fatty alcohol. In some embodiments, the C6-C24 fatty acid can be produced using endogenous enzymes in the host microorganism. In other embodiments, the saturated C6-C24 fatty acid can be produced by one or more exogenous enzymes in the host microorganism.
In some embodiments, the recombinant microorganism disclosed herein includes an acyl-ACP synthetase to catalyze the conversion of a C6-C24 fatty acid to a corresponding saturated C6-C24 fatty acyl-ACP. In some embodiments the acyl-ACP synthetase is a synthetase capable of utilizing a fatty acid as a substrate that has a chain length of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbon atoms. In exemplary embodiments, the recombinant microorganism can include a heterologous the acyl-ACP synthetase from an organism of the species Vibrio harveyi, Rhodotorula glutinis, or Yarrowia lipolytica.
In some embodiments, the recombinant microorganism includes a fatty acyl-ACP desaturase. In some embodiments, the fatty acyl-ACP desaturase is a soluble desaturase. In other embodiments, the fatty-acyl-ACP desaturase is from an organism of the species Pelargonium hortorum, Asclepias syriaca, or Uncaria tomentosa.
In some embodiments, the recombinant microorganism includes a fatty acid synthase complex. In some embodiments, the one or more nucleic acid molecules encoding the fatty acid synthase complex are endogenous nucleic acid molecules. In other embodiments, the one or more nucleic acid molecules encoding a fatty acid synthase complex are exogenous nucleic acid molecules.
In some embodiments, the recombinant microorganism disclosed herein includes a fatty aldehyde forming fatty-acyl reductase which catalyzes the conversion of a C6-C24 fatty acyl-ACP to the corresponding C6-C24 fatty aldehyde. In exemplary embodiments, the fatty aldehyde forming fatty-acyl reductase is from an organism of the species Pelargonium hortorum, Asclepias syriaca, and Uncaria tomentosa. In some embodiments, the recombinant microorganism includes a dehydrogenase to convert the unsaturated fatty aldehyde to a corresponding unsaturated fatty alcohol. In some embodiments, the nucleic acid molecule encoding the dehydrogenase is endogenous to the recombinant microorganism. In other embodiments, the nucleic acid molecule encoding a dehydrogenase is exogenous to the recombinant microorganism. In exemplary embodiments, the endogenous or exogenous nucleic acid molecule encoding a dehydrogenase is isolated from organisms of the species Saccharomyces cerevisiae, Escherichia coli, Yarrowia lipolytica, or Candida tropicalis.
As discussed above, in a third aspect, the application relates to a recombinant microorganism capable of producing an unsaturated C6-C24 fatty alcohol from an endogenous or exogenous source of C6-C24 fatty acid. An illustrative embodiment of the second aspect is shown in
The fatty alcohols produced as taught herein can be further converted to produce downstream products such as insect pheromones, fragrances, flavors, and polymer intermediates, which utilize aldehydes or acetate functional groups. Thus, in some embodiments, the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an alcohol oxidase or an alcohol dehydrogenase, wherein the alcohol oxidase or alcohol dehydrogenase is capable of catalyzing the conversion of a C6-C24 fatty alcohol into a corresponding C6-C24 fatty aldehyde. In other embodiments, the recombinant microorganism can further comprise at least one endogenous or exogenous nucleic acid molecule encoding an acetyl transferase capable of catalyzing the conversion of a C6-C24 fatty alcohol into a corresponding C6-C24 fatty acetate. In certain embodiments, the acetyl transferase, or the nucleic acid sequence that encodes it, can be isolated from organisms of the species Candida glabrata, Saccharomyces cerevisiae, Danaus plexippus, Heliotis virescens, Bombyx mori, Agrotis Ipsilon, Agrotis segetum, Euonymus alatus, Homo sapiens, Lachancea thermotolerans and Yarrowia lipolytica. In exemplary embodiments, the acetyl transferase comprises a sequence selected from GenBank Accession Nos. AY242066, AY242065, AY242064, AY242063, AY242062, EHJ65205, ACX53812, NP_001182381, EHJ65977, EHJ68573, KJ579226, GU594061 KTA99184.1, AIN34693.1, AY605053, XP_002552712.1, XP_503024.1, and XP_505595.1.
Recombinant Microorganism
The disclosure provides microorganisms that can be engineered to express various exogenous enzymes.
In various embodiments described herein, the recombinant microorganism is a eukaryotic microorganism. In some embodiments, the eukaryotic microorganism is a yeast. In exemplary embodiments, the yeast is a member of a genus selected from the group consisting of Yarrowia, Candida, Saccharomyces, Pichia, Hansenula, Kluyveromyces, Issatchenkia, Zygosaccharomyces, Debaryomyces, Schizosaccharomyces, Pachysolen, Cryptococcus, Trichosporon, Rhodotorula, and Myxozyma.
The present inventors have discovered that oleaginous yeast, such as Candida and Yarrowia, have a surprisingly high tolerance to the C6-C24 fatty alcohol substrates and products. Accordingly, in one such exemplary embodiment, the recombinant microorganism of the invention is an oleaginous yeast. In further embodiments, the oleaginous yeast is a member of a genus selected from the group consisting of Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon, and Lipomyces. In even further embodiments, the oleaginous yeast is a member of a species selected from Yarrowia lipolytica, Candida tropicalis, Rhodosporidium toruloides, Lipomyces starkey, L. hpoferus, C. revkaufi, C. pulcherrima, C. utilis, Rhodotorula minuta, Trichosporon pullans, T. cutaneum, Cryptococcus curvatus, R. glutinis, and R. graminis.
In some embodiments, the recombinant microorganism is a prokaryotic microorganism. In exemplary embodiments, the prokaryotic microorganism is a member of a genus selected from the group consisting of Escherichia, Clostridium, Zymomonas, Salmonella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, and Brevibacterium.
In some embodiments, the recombinant microorganism is used to produce a mono- or poly-unsaturated C6-C24 fatty alcohol, aldehyde, or acetate disclosed herein.
Accordingly, in another aspect, the present inventions provide a method of producing a mono- or poly-unsaturated C6-C24 fatty alcohol, aldehyde, or acetate using a recombinant microorganism described herein. In one embodiment, the method comprises cultivating the recombinant microorganism in a culture medium containing a feedstock providing a carbon source until the mono- or poly-unsaturated C6-C24 fatty alcohol, aldehyde, or acetate is produced. In some embodiments, the method comprises cultivating the recombinant microorganism described herein in a culture medium containing a feedstock that provides a carbon source adequate for the production of a mono- or poly-unsaturated ≤C18 fatty alcohol, fatty aldehyde or fatty acetate. In a further embodiment, the mono- or poly-unsaturated ≤C18 fatty alcohol, aldehyde, or acetate is recovered. Recovery can be by methods known in the art, such as distillation, membrane-based separation gas stripping, solvent extraction, and expanded bed adsorption.
In some embodiments, the feedstock comprises a carbon source. In various embodiments described herein, the carbon source may be selected from sugars, glycerol, alcohols, organic acids, alkanes, fatty acids, lignocellulose, proteins, carbon dioxide, and carbon monoxide. In a further embodiment, the sugar is selected from the group consisting of glucose, fructose, and sucrose.
Methods of Engineering Microorganisms that are Capable of Producing Mono- or Poly-Unsaturated C6-C24 Fatty Alcohols, Fatty Aldehydes and/or Fatty Acetates
In one aspect, the present disclosure provides a method of engineering a microorganism that is capable of producing a mono- or poly-unsaturated ≤C18 fatty alcohol from an endogenous or exogenous source of saturated C6-C24 fatty acid, wherein the method comprises introducing into a microorganism the following: (a) at least one exogenous nucleic acid molecule encoding a fatty acyl desaturase that catalyzes the conversion of a saturated C6-C24 fatty acyl-CoA to a corresponding mono- or poly-unsaturated C6-C24 fatty acyl-CoA; (b) at least one exogenous nucleic acid molecule encoding an acyl-CoA oxidase that catalyzes the conversion of the mono- or poly-unsaturated C6-C24 fatty acyl-CoA from (a) into a mono- or poly-unsaturated ≤C18 fatty acyl-CoA after one or more successive cycle of acyl-CoA oxidase activity, with a given cycle producing a mono- or poly-unsaturated C4-C22 fatty acyl-CoA intermediate with a two carbon truncation relative to a starting mono- or poly-unsaturated C6-C24 fatty acyl-CoA substrate in that cycle; and (c) at least one exogenous nucleic acid molecule encoding a fatty alcohol forming fatty acyl reductase that catalyzes the conversion of the mono- or poly-unsaturated ≤C18 fatty acyl-CoA from (b) into the corresponding mono- or poly-unsaturated ≤C18 fatty alcohol. In some embodiments, the microorganism is MATA ura3-302::SUC2 Δpox1 Δpox2 Δpox3 Δpox4 Δpox5 Δpox6 Δfadh Δadh1 Δadh2 Δadh3 Δadh4 Δadh5 Δadh6 Δadh7 Δfao1::URA3.
In some embodiments, the fatty acyl desaturase is selected from an Argyrotaenia velutinana, Spodoptera litura, Sesamia inferens, Manduca sexta, Ostrinia nubilalis, Helicoverpa zea, Choristoneura rosaceana, Drosophila melanogaster, Spodoptera littoralis, Lampronia capitella, Amyelois transitella, Trichoplusia ni, Agrotis segetum, Ostrinia furnicalis, and Thalassiosira pseudonana derived fatty acyl desaturase. In some preferred embodiments, the fatty acyl desaturase has 95% sequence identity to a fatty acyl desaturase selected from the group consisting of: SEQ ID NOs: 39, 49-54, 58-63, 78-80 and GenBank Accession nos. AF416738, AGH12217.1, AII21943.1, CAJ43430.2, AF441221, AAF81787.1, AF545481, AJ271414, AY362879, ABX71630.1 and NP001299594.1, Q9N9Z8, ABX71630.1 and AIM40221.1. In further embodiments, the acyl-CoA oxidase is selected from Table 5a. In yet further embodiments, the fatty alcohol forming fatty acyl reductase is selected from an Agrotis segetum, Spodoptera exigua, Spodoptera littoralis, Euglena gracilis, Yponomeuta evonymellus and Helicoverpa armigera derived fatty alcohol forming fatty acyl reductase. In further embodiments, the fatty alcohol forming fatty acyl reductase has 90% sequence identity to a fatty alcohol forming fatty acyl reductase selected from the group consisting of: SEQ ID NOs: 1-3, 32, 41-48, 55-57, 73, 75, 77 and 82. In some embodiments, the recombinant microorganism is a yeast selected from the group consisting of Yarrowia lipolytica, Saccharomyces cerevisiae, Candida albicans, Candida tropicalis and Candida viswanathii.
In some embodiments, the fatty acyl desaturase catalyzes the conversion of a fatty acyl-CoA into a mono- or poly-unsaturated intermediate selected from E5-10:Acyl-CoA, E7-12:Acyl-CoA, E9-14:Acyl-CoA, E11-16:Acyl-CoA, E13-18:Acyl-CoA, Z7-12:Acyl-CoA, Z9-14:Acyl-CoA, Z11-16:Acyl-CoA, Z13-18:Acyl-CoA, Z8-12:Acyl-CoA, Z10-14:Acyl-CoA, Z12-16:Acyl-CoA, Z14-18:Acyl-CoA, Z7-10:Acyl-coA, Z9-12:Acyl-CoA, Z11-14:Acyl-CoA, Z13-16:Acyl-CoA, Z15-18:Acyl-CoA, E7-10:Acyl-CoA, E9-12:Acyl-CoA, E11-14:Acyl-CoA, E13-16:Acyl-CoA, E15-18:Acyl-CoA, E5Z7-12:Acyl-CoA, E7Z9-12:Acyl-CoA, E9Z11-14:Acyl-CoA, E11Z13-16:Acyl-CoA, E13Z15-18:Acyl-CoA, E6E8-10:Acyl-CoA, E8E10-12:Acyl-CoA, E10E12-14:Acyl-CoA, E12E14-16:Acyl-CoA, Z5E8-10:Acyl-CoA, Z7E10-12:Acyl-CoA, Z9E12-14:Acyl-CoA, Z11E14-16:Acyl-CoA, Z13E16-18:Acyl-CoA, Z3-10:Acyl-CoA, Z5-12:Acyl-CoA, Z7-14:Acyl-CoA, Z9-16:Acyl-CoA, Z11-18:Acyl-CoA, Z3Z5-10:Acyl-CoA, Z5Z7-12:Acyl-CoA, Z7Z9-14:Acyl-CoA, Z9Z11-16:Acyl-CoA, Z11Z13-16:Acyl-CoA, and Z13Z15-18:Acyl-CoA. In further embodiments, the mono- or poly-unsaturated ≤C18 fatty alcohol is selected from the group consisting of E5-10:OH, Z8-12:OH, Z9-12:OH, Z11-14:OH, Z11-16:OH, E11-14:OH, E8E10-12:OH, E7Z9-12:OH, Z11Z13-16OH, Z9-14:OH, Z9-16:OH, and Z13-18:OH.
In some embodiments, the method further comprises introducing into the microorganism at least one endogenous or exogenous nucleic acid molecule encoding an acyltransferase that preferably stores ≤C18 fatty acyl-CoA. In some embodiments, the acyltransferase is selected from the group consisting of glycerol-3-phosphate acyl transferase (GPAT), lysophosphatidic acid acyltransferase (LPAAT), glycerolphospholipid acyltransferase (GPLAT) and diacylglycerol acyltransferases (DGAT). In some preferred embodiments, the acyltransferase is selected from Table 5b.
In some embodiments, the method further comprises introducing into the microorganism at least one endogenous or exogenous nucleic acid molecule encoding an acylglycerol lipase that preferably hydrolyzes ester bonds of >C16, of >C14, of >C12 or of >C10 acylglycerol substrates. In some embodiments, the acylglycerol lipase is selected from Table 5c.
In some embodiments, the method further comprises introducing into the microorganism one or more modifications comprising a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous enzyme that catalyzes a reaction in a pathway that competes with the biosynthesis pathway for the production of a mono- or poly-unsaturated ≤C18 fatty alcohol. In further embodiments, the recombinant microorganism comprises a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous enzyme selected from: (i) one or more acyl-CoA oxidase; (ii) one or more acyltransferase; (iii) one or more acylglycerol lipase and/or sterol ester esterase; (iv) one or more (fatty) alcohol dehydrogenase; (v) one or more (fatty) alcohol oxidase; and (vi) one or more cytochrome P450 monooxygenase.
In some embodiments, the method further comprises introducing into the microorganism one or more modifications comprising a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous acyl-CoA oxidase enzyme selected from the group consisting of Y. lipolytica POX1(YALI0E32835g), Y. lipolytica POX2(YALI0F10857g), Y. lipolytica POX3(YALI0D24750g), Y. lipolytica POX4(YALI0E27654g), Y. lipolytica POX5(YALI0C23859g), Y. lipolytica POX6(YALI0E06567g); S. cerevisiae POX1(YGL205W); Candida POX2 (CaO19.1655, CaO19.9224, CTRG_02374, M18259), Candida POX4 (CaO19.1652, CaO19.9221, CTRG_02377, M12160), and Candida POX5 (CaO19.5723, CaO19.13146, CTRG_02721, M12161).
In some embodiments, the method further comprises introducing into the microorganism one or more modifications comprising a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous acyltransferase enzyme selected from the group consisting of Y. lipolytica YALI0C00209g, Y. lipolytica YALI0E18964g, Y. lipolytica YALI0F19514g, Y. lipolytica YALI0C14014g, Y. lipolytica YALI0E16797g, Y. lipolytica YALI0E32769g, and Y. lipolytica YALI0D07986g, S. cerevisiae YBL011w, S. cerevisiae YDL052c, S. cerevisiae YOR175C, S. cerevisiae YPR139C, S. cerevisiae YNR008w, and S. cerevisiae YOR245c, and Candida I503_02577, Candida CTRG_02630, Candida CaO19.250, Candida CaO19.7881, Candida CTRG_02437, Candida CaO19.1881, Candida CaO19.9437, Candida CTRG_01687, Candida CaO19.1043, Candida CaO19.8645, Candida CTRG_04750, Candida CaO19.13439, Candida CTRG_04390, Candida CaO19.6941, Candida CaO19.14203, and Candida CTRG_06209.
In some embodiments, the method further comprises introducing into the microorganism one or more modifications comprising a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous acylglycerol lipase and/or sterol ester esterase enzyme selected from the group consisting of Y. lipolytica YALI0E32035g, Y. lipolytica YALI0D17534g, Y. lipolytica YALI0F10010g, Y. lipolytica YALI0C14520g, and Y. lipolytica YALI0E00528g, S. cerevisiae YKL140w, S. cerevisiae YMR313c, S. cerevisiae YKR089c, S. cerevisiae YOR081c, S. cerevisiae YKL094W, S. cerevisiae YLL012W, and S. cerevisiae YLR020C, and Candida CaO19.2050, Candida CaO19.9598, Candida CTRG_01138, Candida W5Q_03398, Candida CTRG_00057, Candida CaO19.5426, Candida CaO19.12881, Candida CTRG_06185, Candida CaO19.4864, Candida CaO19.12328, Candida CTRG_03360, Candida CaO19.6501, Candida CaO19.13854, Candida CTRG_05049, Candida CaO19.1887, Candida CaO19.9443, Candida CTRG_01683, and Candida CTRG_04630.
In some embodiments, the method further comprises introducing into the microorganism one or more modifications comprising a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous cytochrome P450 monooxygenases selected from the group consisting of Y. lipolytica YALI0E25982g (ALK1), Y. lipolytica YALI0F01320g (ALK2), Y. lipolytica YALI0E23474g (ALK3), Y. lipolytica YALI0B13816g (ALK4), Y. lipolytica YALI0B13838g (ALK5), Y. lipolytica YALI0B01848g (ALK6). Y. lipolytica YALI0A15488g (ALK7), Y. lipolytica YALI0C12122g (ALK8), Y. lipolytica YALI0B06248g (ALK9), Y. lipolytica YALI0B20702g (ALK0), Y. lipolytica YALI0C10054g (ALK11) and Y. lipolytica YALI0A20130g (ALK12).
In some embodiments, the method further comprises introducing into the microorganism at least one endogenous or exogenous nucleic acid molecule encoding an aldehyde forming fatty acyl-CoA reductase capable of catalyzing the conversion of the mono- or poly-unsaturated ≤C18 fatty alcohol into a corresponding ≤C18 fatty aldehyde. In some preferred embodiments, the aldehyde forming fatty acyl-CoA reductase is selected from the group consisting of Acinetobacter calcoaceticus A0A1C4HN78, A. calcoaceticus N9DA85, A. calcoaceticus R8XW24, A. calcoaceticus A0A1A0GGM5, A. calcoaceticus A0A117N158, and Nostoc punctiforme YP_001865324. In some embodiments, the method further comprises introducing into the microorganism at least one endogenous or exogenous nucleic acid molecule encoding an alcohol oxidase or an alcohol dehydrogenase capable of catalyzing the conversion of the mono- or poly-unsaturated ≤C18 fatty alcohol into a corresponding ≤C18 fatty aldehyde. In some preferred embodiments, the ≤C18 fatty aldehyde is selected from the group consisting of Z9-16:Ald, Z11-16:Ald, Z11Z13-16:Ald, and Z13-18:Ald.
In some embodiments, the method further comprises introducing into the microorganism at least one endogenous or exogenous nucleic acid molecule encoding an acetyl transferase capable of catalyzing the conversion of the mono- or poly-unsaturated ≤C18 fatty alcohol into a corresponding ≤C18 fatty acetate. In some embodiments, the acetyl transferase is selected from Table 5d. In some preferred embodiments, the ≤C18 fatty acetate is selected from the group consisting of E5-10:Ac, Z7-12:Ac, Z8-12:Ac, Z9-12:Ac, E7Z9-12:Ac, Z9-14:Ac, Z9E12-14:Ac, Z11-14:Ac, E11-14:Ac, Z9-16:Ac, and Z11-16:Ac.
In some embodiments, the method further comprises introducing into the microorganism: at least one endogenous or exogenous nucleic acid molecule encoding an enzyme selected from an alcohol oxidase, an alcohol dehydrogenase, and an aldehyde forming fatty acyl-CoA reductase capable of catalyzing the conversion of the mono- or poly-unsaturated ≤C18 fatty alcohol into a corresponding ≤C18 fatty aldehyde; and at least one endogenous or exogenous nucleic acid molecule encoding an acetyl transferase capable of catalyzing the conversion of the mono- or poly-unsaturated ≤C18 fatty alcohol into a corresponding ≤C18 fatty acetate. In some preferred embodiments, the mono- or poly-unsaturated ≤C18 fatty aldehyde and ≤C18 fatty acetate is selected from the group consisting of E5-10:Ac, Z7-12:Ac, Z8-12:Ac, Z9-12:Ac, E7Z9-12:Ac, Z9-14:Ac, Z9E12-14:Ac, E11-14:Ac, Z11-14:Ac, Z11-16:Ac, Z9-16:Ac, Z9-16:Ald, Z11-16:Ald, Z11Z13-16:Ald, and Z13-18:Ald.
In some embodiments, the disclosure provides a method of producing a mono- or poly-unsaturated ≤C18 fatty alcohol, fatty aldehyde or fatty acetate from an endogenous or exogenous source of saturated C6-C24 fatty acid, comprising: cultivating a recombinant microorganism described herein in a culture medium containing a feedstock that provides a carbon source adequate for the production of the mono- or poly-unsaturated ≤C18 fatty alcohol, fatty aldehyde or fatty acetate. In some embodiments, the method further comprises a step of recovering the mono- or poly-unsaturated ≤C18 fatty alcohol, fatty aldehyde or fatty acetate. In further embodiments, the recovery step comprises distillation. In yet further embodiments, the recovery step comprises membrane-based separation.
In some embodiments, the mono- or poly-unsaturated ≤C18 fatty alcohol is converted into a corresponding ≤C18 fatty aldehyde using chemical methods. In further embodiments, the chemical methods are selected from TEMPO-bleach, TEMPO-copper-air, TEMPO-PhI(OAc)2, Swern oxidation and noble metal-air. In some embodiments, the mono- or poly-unsaturated ≤C18 fatty alcohol is converted into a corresponding ≤C18 fatty acetate using chemical methods. In further embodiments, the chemical methods utilize a chemical agent selected from the group consisting of acetyl chloride, acetic anhydride, butyryl chloride, butyric anhydride, propanoyl chloride and propionic anhydride in the presence of 4-N,N-dimethylaminopyridine (DMAP) or sodium acetate to esterify the mono- or poly-unsaturated ≤C18 fatty alcohol to the corresponding ≤C18 fatty acetate.
In another aspect, the present disclosure provides methods of engineering a Yarrowia lipolytica microorganism capable of producing a mono- or poly-unsaturated C6-C24 fatty alcohol from an endogenous or exogenous source of saturated C6-C24 fatty acid, wherein the recombinant Yarrowia lipolytica microorganism comprises: (a) at least one nucleic acid molecule encoding a fatty acyl desaturase having 95% sequence identity to a fatty acyl desaturase selected from the group consisting of SEQ ID NOs: 54, 60, 62, 78, 79, 80, 95, 97, 99, 101, 103, and 105 that catalyzes the conversion of a saturated C6-C24 fatty acyl-CoA to a corresponding mono- or poly-unsaturated C6-C24 fatty acyl-CoA; and (b) at least one nucleic acid molecule encoding a fatty alcohol forming fatty acyl reductase having 95% sequence identity to a fatty alcohol forming fatty acyl reductase selected from the group consisting of SEQ ID NOs: 41-48, 57, 73, 75 and 77 that catalyzes the conversion of the mono- or poly-unsaturated C6-C24 fatty acyl-CoA from (a) into the corresponding mono- or poly-unsaturated C6-C24 fatty alcohol.
In some embodiments, the method further comprises introducing into the Yarrowia lipolytica microorganism one or more modifications comprising a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous enzymes that catalyzes a reaction in a pathway that competes with the biosynthesis pathway for the production of a mono- or poly-unsaturated C6-C24 fatty alcohol. In some preferred embodiments, the recombinant Yarrowia lipolytica microorganism comprises a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous enzyme selected from the following: (i) one or more acyl-CoA oxidase selected from the group consisting of YALI0E32835g (POX1), YALI0F10857g (POX2), YALI0D24750g (POX3), YALI0E27654g (POX4), YALI0C23859g (POX5), YALI0E06567g (POX6); (ii) one or more (fatty) alcohol dehydrogenase selected from the group consisting of YALI0F09603g (FADH), YALI0D25630g (ADH1), YALI0E17787g (ADH2), YALI0A16379g (ADH3), YALI0E15818g (ADH4), YALI0D02167g (ADH5), YALI0A15147g (ADH6), YALI0E07766g (ADH7); (iii) a (fatty) alcohol oxidase YALI0B14014g (FAO1); (iv) one or more cytochrome P450 enzyme selected from the group consisting of YALI0E25982g (ALK1), YALI0F01320g (ALK2), YALI0E23474g (ALK3), YALI0B13816g (ALK4), YALI0B13838g (ALK5), YALI0B01848g (ALK6), YALI0A15488g (ALK7), (YALI0C12122g (ALK8), YALI0B06248g (ALK9), YALI0B20702g (ALK10), YALI0C10054g (ALK11) and YALI0A20130g (Alk12); and (v) one or more diacylglycerol acyltransferase selected from the group consisting of YALI0E32791g (DGA1) and YALI0D07986g (DGA2). In other preferred embodiments, the recombinant Yarrowia lipolytica microorganism comprises a deletion of one or more endogenous enzyme selected from the following: (i) one or more acyl-CoA oxidase selected from the group consisting of YALI0E32835g (POX1), YALI0F10857g (POX2), YALI0D24750g (POX3), YALI0E27654g (POX4), YALI0C23859g (POX5), YALI0E06567g (POX6); (ii) one or more (fatty) alcohol dehydrogenase selected from the group consisting of YALI0F09603g (FADH), YALI0D25630g (ADH1), YALI0E17787g (ADH2), YALI0A16379g (ADH3), YALI0E15818g (ADH4), YALI0D02167g (ADH5), YALI0A15147g (ADH6), YALI0E07766g (ADH7); (iii) a (fatty) alcohol oxidase YALI0B14014g (FAO1); (iv) one or more cytochrome P450 enzyme selected from the group consisting of YALI0E25982g (ALK1), YALI0F01320g (ALK2), YALI0E23474g (ALK3), YALI0B13816g (ALK4), YALI0B13838g (ALK5), YALI0B01848g (ALK6), YALI0A15488g (ALK7), (YALI0C12122g (ALK8), YALI0B06248g (ALK9), YALI0B20702g (ALK10), YALI0C10054g (ALK11) and YALI0A20130g (Alk12); and (v) one or more diacylglycerol acyltransferase selected from the group consisting of YALI0E32791g (DGA1) and YALI0D07986g (DGA2).
In some embodiments, the fatty acyl desaturase catalyzes the conversion of a saturated fatty acyl-CoA into a mono- or poly-unsaturated intermediate selected from Z9-14:Acyl-CoA, Z11-14:Acyl-CoA, E11-14:Acyl-CoA, Z9-16:Acyl-CoA, and Z11-16:Acyl-CoA. In other embodiments, the mono- or poly-unsaturated C6-C24 fatty alcohol is selected from the group consisting of Z9-14:OH, Z11-14:OH, E11-14:OH, Z9-16:OH, Z11-16:OH, Z11Z13-16:OH, and Z13-18:OH.
In some embodiments, the method further comprises introducing into the Yarrowia lipolytica microorganism at least one endogenous or exogenous nucleic acid molecule encoding an alcohol oxidase or an alcohol dehydrogenase capable of catalyzing the conversion of the mono- or poly-unsaturated C6-C24 fatty alcohol into a corresponding C6-C24 fatty aldehyde. In some embodiments, the alcohol dehydrogenase is selected from Table 3a. In some embodiments, the C6-C24 fatty aldehyde is selected from the group consisting of Z9-14:Ald, Z11-14:Ald, E11-14:Ald, Z9-16:Ald, Z11-16:Ald, Z11Z13-16:Ald and Z13-18:Ald.
In some embodiments, the method further comprises introducing into the Yarrowia lipolytica microorganism at least one endogenous or exogenous nucleic acid molecule encoding an acetyl transferase capable of catalyzing the conversion of the mono- or poly-unsaturated C6-C24 fatty alcohol into a corresponding C6-C24 fatty acetate. In some embodiments, the acetyl transferase is selected from Table 5d. In some embodiments, the C6-C24 fatty acetate is selected from the group consisting of Z9-14:Ac, Z11-14:Ac, E11-14:Ac, Z9-16:Ac, Z11-16:Ac, Z11Z13-16:Ac, and Z13-18:Ac.
In some embodiments, the method further comprises introducing into the Yarrowia lipolytica microorganism: at least one endogenous or exogenous nucleic acid molecule encoding an alcohol oxidase or an alcohol dehydrogenase capable of catalyzing the conversion of the mono- or poly-unsaturated C6-C24 fatty alcohol into a corresponding C6-C24 fatty aldehyde; and at least one endogenous or exogenous nucleic acid molecule encoding an acetyl transferase capable of catalyzing the conversion of the mono- or poly-unsaturated C6-C24 fatty alcohol into a corresponding C6-C24 fatty acetate. In some embodiments, the mono- or poly-unsaturated C6-C24 fatty aldehyde and C6-C24 fatty acetate is selected from the group consisting of Z9-14:Ac, Z11-14:Ac, E11-14:Ac, Z9-16:Ac, Z11-16:Ac, Z11Z13-16:Ac, Z13-18:Ac, Z9-14:Ald, Z11-14:Ald, E11-14:Ald, Z9-16:Ald, Z11-16:Ald, Z11Z13-16:Ald and Z13-18:Ald.
In some embodiments, the fatty acyl desaturase does not comprise a fatty acyl desaturase comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 64, 65, 66 and 67. In other embodiments, the fatty acyl desaturase does not comprise a fatty acyl desaturase selected from an Amyelois transitella, Spodoptera littoralis, Agrotis segetum, or Trichoplusia ni derived desaturase.
In some embodiments, the disclosure provides a method of producing a mono- or poly-unsaturated C6-C24 fatty alcohol, fatty aldehyde or fatty acetate from an endogenous or exogenous source of saturated C6-C24 fatty acid, comprising: cultivating a recombinant microorganism described herein in a culture medium containing a feedstock that provides a carbon source adequate for the production of the mono- or poly-unsaturated C6-C24 fatty alcohol, fatty aldehyde or fatty acetate. In some embodiments, the method further comprises a step of recovering the mono- or poly-unsaturated C6-C24 fatty alcohol, fatty aldehyde or fatty acetate. In further embodiments, the recovery step comprises distillation. In yet further embodiments, the recovery step comprises membrane-based separation.
In some embodiments, the mono- or poly-unsaturated C6-C24 fatty alcohol is converted into a corresponding C6-C24 fatty aldehyde using chemical methods. In further embodiments, the chemical methods are selected from TEMPO-bleach, TEMPO-copper-air, TEMPO-PhI(OAc)2, Swern oxidation and noble metal-air. In some embodiments, the mono- or poly-unsaturated C6-C24 fatty alcohol is converted into a corresponding C6-C24 fatty acetate using chemical methods. In further embodiments, the chemical methods utilize a chemical agent selected from the group consisting of acetyl chloride, acetic anhydride, butyryl chloride, butyric anhydride, propanoyl chloride and propionic anhydride in the presence of 4-N,N-dimethylaminopyridine (DMAP) or sodium acetate to esterify the mono- or poly-unsaturated C6-C24 fatty alcohol to the corresponding C6-C24 fatty acetate.
Enzyme Engineering
The enzymes in the recombinant microorganism can be engineered to improve one or more aspects of the substrate to product conversion. Non-limiting examples of enzymes that can be further engineered for use in methods of the disclosure include a desaturase (e.g., a fatty acyl-CoA desaturase or fatty acyl-ACP desaturase), a fatty alcohol forming fatty acyl reductase, an acyl-ACP synthetase, a fatty acid synthetase, a fatty acid synthase complex, an acetyl transferase, dehydrogenase, and an alcohol oxidase, and combinations thereof. These enzymes can be engineered for improved catalytic activity, improved selectivity, improved stability, improved tolerance to various fermentations conditions (temperature, pH, etc.), or improved tolerance to various metabolic substrates, products, by-products, intermediates, etc.
Desaturase enzymes can be engineered for improved catalytic activity in the desaturation of an unsaturated substrate, for improved hydrocarbon selectivity, for improved selectivity of a Z product over an E product, or an E product over a Z product. For example, the Z9 fatty-acyl desaturase can be engineered to improve the yield in the substrate to product conversion of a saturated fatty acyl-CoA to the corresponding unsaturated fatty acyl-CoA, and, in addition or in the alternative, to improve selectivity of the desaturation at the 9 position to produce a corresponding Z-9 fatty acyl-CoA. In further non-limiting examples, the fatty acyl-ACP synthetase can be engineered for improved ACP ligation activity; a fatty acid synthase complex enzyme can be engineered for improved catalytic activity of elongation of a fatty acid substrate; a fatty alcohol forming fatty acyl-reductase can be engineered for improved catalytic activity in the reduction of a fatty acyl-CoA to a corresponding fatty alcohol; a fatty aldehyde forming fatty acyl-reductase can be engineered for improved catalytic activity in the reduction of a fatty acyl-ACP to a corresponding fatty aldehyde; a dehydrogenase can be engineered for improved catalytic activity in the conversion of a fatty acyl-ACP to a corresponding fatty alcohol; an alcohol oxidase can be engineered for improved catalytic activity in the conversion of a fatty alcohol into a corresponding fatty aldehyde; and an acetyl transferase can be engineered for improved catalytic activity in the conversion of a fatty alcohol into a corresponding fatty acetate.
The term “improved catalytic activity” as used herein with respect to a particular enzymatic activity refers to a higher level of enzymatic activity than that measured relative to a comparable non-engineered enzyme, such as a non-engineered desaturase (e.g. fatty acyl-CoA desaturase or fatty acyl-ACP desaturase), fatty alcohol or aldehyde forming fatty-acyl reductase, acyl-ACP synthetase, fatty acid synthetase, fatty acid synthase complex, acyl transferase, dehydrogenase, or an alcohol oxidase enzyme. For example, overexpression of a specific enzyme can lead to an increased level of activity in the cells for that enzyme. Mutations can be introduced into a desaturase (e.g. fatty acyl-CoA desaturase or fatty acyl-ACP desaturase), a fatty alcohol or aldehyde forming fatty-acyl reductase, a acyl-ACP synthetase, a fatty acid synthetase, a fatty acid synthase complex, a acyl transferase, a dehydrogenase, or an alcohol oxidase enzyme resulting in engineered enzymes with improved catalytic activity. Methods to increase enzymatic activity are known to those skilled in the art. Such techniques can include increasing the expression of the enzyme by increasing plasmid copy number and/or use of a stronger promoter and/or use of activating riboswitches, introduction of mutations to relieve negative regulation of the enzyme, introduction of specific mutations to increase specific activity and/or decrease the KM for the substrate, or by directed evolution. See, e.g., Methods in Molecular Biology (vol. 231), ed. Arnold and Georgiou, Humana Press (2003).
Metabolic Engineering—Enzyme Overexpression and Gene Deletion/Downregulation for Increased Pathway Flux
In various embodiments described herein, the exogenous and endogenous enzymes in the recombinant microorganism participating in the biosynthesis pathways described herein may be overexpressed.
The terms “overexpressed” or “overexpression” refers to an elevated level (e.g., aberrant level) of mRNAs encoding for a protein(s), and/or to elevated levels of protein(s) in cells as compared to similar corresponding unmodified cells expressing basal levels of mRNAs or having basal levels of proteins. In particular embodiments, mRNA(s) or protein(s) may be overexpressed by at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 8-fold, 10-fold, 12-fold, 15-fold or more in microorganisms engineered to exhibit increased gene mRNA, protein, and/or activity.
In some embodiments, a recombinant microorganism of the disclosure is generated from a host that contains the enzymatic capability to synthesize a substrate fatty acid. In this specific embodiment it can be useful to increase the synthesis or accumulation of a fatty acid to, for example, increase the amount of fatty acid available to an engineered fatty alcohol production pathway.
In some embodiments, it may be useful to increase the expression of endogenous or exogenous enzymes involved in the fatty alcohol, aldehyde, or acetate production pathway to increase flux from the fatty acid to the fatty alcohol, aldehyde, or acetate, thereby resulting in increased synthesis or accumulation of the fatty alcohol, aldehyde, or acetate.
In some embodiments, it may be useful to increase the expression of endogenous or exogenous enzymes to increase intracellular levels of a coenzyme. In one embodiment, the coenzyme is NADH. In another embodiment, the coenzyme is NADPH. In one embodiment, the expression of proteins in the pentose phosphate pathway is increased to increase the intracellular levels of NADPH. The pentose phosphate pathway is an important catabolic pathway for supplying reduction equivalents and an important anabolic pathway for biosynthesis reactions. In one embodiment, a glucose-6-phosphate dehydrogenase that converts glucose-6-phosphate to 6-phospho D-glucono-1,5-lactone is overexpressed. In some embodiments, the glucose-6-phosphate dehydrogenase is ZWF1 from yeast. In another embodiment, the glucose-6-phosphate dehydrogenase is ZWF1 (YNL241C) from Saccharomyces cerevisiae. In one embodiment, a glucose-6-phosphate-1-dehydrogenase that converts D-glucopyranose-6-phosphate to 6-phospho D-glucono-1,5-lactone is overexpressed. In another embodiment, the glucose-6-phosphate-1-dehydrogenase is zwf from bacteria. In certain embodiments, the glucose-6-phosphate-1-dehydrogenase is zwf (NP_416366) from E. coli. In one embodiment, a 6-phosphogluconolactonase that converts 6-phospho D-glucono-1,5-lactone to D-gluconate 6-phosphate is overexpressed. In some embodiments, the 6-phosphogluconolactonase is SOL3 of yeast. In certain embodiments, the 6-phosphogluconolactonase is SOL3 (NP_012033) of Saccharomyces cerevisiae. In some embodiments, the 6-phosphogluconolactonase is SOL4 of yeast. In certain embodiments, the 6-phosphogluconolactonase is SOL4 (NP_011764) of Saccharomyces cerevisiae. In some embodiments, the 6-phosphogluconolactonase is pgl of bacteria. In certain embodiments, the 6-phosphogluconolactonase is pgl (NP_415288) of E. coli. In one embodiment, a 6-phosphogluconate dehydrogenase that converts D-gluconate 6-phosphate to D-ribulose 5-phosphate is overexpressed. In some embodiments, the 6-phosphogluconate dehydrogenase is GND1 from yeast. In certain embodiments, the 6-phosphogluconate dehydrogenase is GND1 (YHR183W) from Saccharomyces cerevisiae. In some embodiments, the 6-phosphogluconate dehydrogenase is GND2 from yeast. In certain embodiments, the 6-phosphogluconate dehydrogenase is GND2 (YGR256W) from Saccharomyces cerevisiae. In some embodiments, the 6-phosphogluconate dehydrogenase is gnd from bacteria. In certain embodiments, the 6-phosphogluconate dehydrogenase is gnd (NP_416533) from E. coli. In one embodiment, a transaldolase that interconverts D-glyceraldehyde 3-phosphate and D-sedoheptulose 7-phosphate to β-D-fructofuranose 6-phosphate and D-erythrose 4-phosphate is overexpressed. In some embodiments, the transaldolase is TAL1 of yeast. In certain embodiments, the transaldolase is TAL1 (NP_013458) of Saccharomyces cerevisiae. In some embodiments, the transaldolase is NQM1 of yeast. In certain embodiments, the transaldolase is NQM1 (NP_011557) of Saccharomyces cerevisiae. In some embodiments, the transaldolase is tal of bacteria. In certain embodiments, the transaldolase is talB (NP_414549) of E. coli. In certain embodiments, the transaldolase is talA (NP_416959) of E. coli. In one embodiment, a transketolase that interconverts D-erythrose 4-phosphate and D-xylulose 5-phosphate to β-D-fructofuranose 6-phosphate and D-glyceraldehyde 3-phosphate and/or interconverts D-sedoheptulose 7-phosphate and D-glyceraldehyde 3-phosphate to D-ribose 5-phosphate and D-xylulose 5-phosphate is overexpressed. In some embodiments, the transketolase is TKL1 of yeast. In certain embodiments, the transketolase is TKL1 (NP_015399) of Saccharomyces cerevisiae. In some embodiments, the transketolase is TKL2 of yeast. In some embodiments, the transketolase is TKL2 (NP_009675) of Saccharomyces cerevisiae. In some embodiments, the transketolase is tkt of bacteria. In certain embodiments, the transketolase is tktA (YP_026188) of E. coli. In certain embodiments, the transketolase is tktB (NP_416960) of E. coli. In one embodiment, a ribose-5-phosphate ketol-isomerase that interconverts D-ribose 5-phosphate and D-ribulose 5-phosphate is overexpressed. In some embodiments, the ribose-5-phosphate ketol-isomerase is RKI1 of yeast. In certain embodiments, the ribose-5-phosphate ketol-isomerase is RKI1 (NP_014738) of Saccharomyces cerevisiae. In some embodiments, the ribose-5-phosphate isomerase is rpi of bacteria. In certain embodiments, the ribose-5-phosphate isomerase is rpiA (NP_417389) of E. coli. In certain embodiments, the ribose-5-phosphate isomerase is rpiB (NP_418514) of E. coli. In one embodiment, a D-ribulose-5-phosphate 3-epimerase that interconverts D-ribulose 5-phosphate and D-xylulose 5-phosphate is overexpressed. In some embodiments, the D-ribulose-5-phosphate 3-epimerase is RPE1 of yeast. In certain embodiments, the D-ribulose-5-phosphate 3-epimerase is RPE1 (NP_012414) of Saccharomyces cerevisiae. In some embodiments, the D-ribulose-5-phosphate 3-epimerase is rpe of bacteria. In certain embodiments, the D-ribulose-5-phosphate 3-epimerase is rpe (NP_417845) of E. coli.
In one embodiment, the expression of an NADP+-dependent isocitrate dehydrogenase is increased to increase intracellular levels of a coenzyme. In one embodiment, an NADP+ dependent isocitrate dehydrogenase oxidizes D-threo-isocitrate to 2-oxoglutarate with concomitant generation of NADPH. In another embodiment, an NADP+ dependent isocitrate dehydrogenase oxidizes D-threo-isocitrate to 2-oxalosuccinate with concomitant generation of NADPH. In some embodiments, the NADP+-dependent isocitrate dehydrogenase is IDP from yeast. In certain embodiments, the NADP+-dependent isocitrate dehydrogenase is IDP2 (YLR174W) from Saccharomyces cerevisiae. In some embodiments, the NADP+-dependent isocitrate dehydrogenase is icd from bacteria. In certain embodiments, the NADP+-dependent isocitrate dehydrogenase is icd (NP_415654) from E. coli.
In some embodiments, the expression of a malic enzyme that decarboxylates malate to pyruvate with concomitant generation of NADH or NADPH is increased to increase intracellular levels of a coenzyme. In one embodiment, the malic enzyme is NAD+ dependent. In another embodiment, the malic enzyme is NADP+ dependent. In one embodiment, the malic enzyme is an NAD+ dependent malate dehydrogenase from bacteria. In some embodiments, the NAD+ dependent malate dehydrogenase is maeA (NP_415996) from E. coli. In some embodiments, the NAD+ dependent malate dehydrogenase is maeE (CAQ68119) from Lactobacillus casei. In another embodiment, the malic enzyme is a mitochondrial NAD+ dependent malate dehydrogenase from yeast. In some embodiments, the NAD+ dependent malate dehydrogenase is MAE1 (YKL029C) from S. cerevisiae. In another embodiment, the malic enzyme is a mitochondrial NAD+ dependent malate dehydrogenase from a parasitic nematode. In some embodiments, the NAD+ dependent malate dehydrogenase is M81055 from Ascaris suum. In one embodiment, the malic enzyme is an NADP+ dependent malate dehydrogenase from bacteria. In some embodiments, the NADP+ dependent malate dehydrogenase is maeB (NP_416958) from E. coli. In one embodiment, the malic enzyme is an NADP+ dependent malate dehydrogenase from corn. In some embodiments, the NADP+ dependent malate dehydrogenase is mel from Zea mays.
In some embodiments, the expression of an aldehyde dehydrogenase that oxidizes an aldehyde to a carboxylic acid with concomitant generation of NADH or NADPH is increased to increase intracellular levels of a coenzyme. In one embodiment, the aldehyde dehydrogenase is NAD+ dependent. In another embodiment, the aldehyde dehydrogenase is NADP+ dependent. In one embodiment, the aldehyde dehydrogenase is an NAD+ dependent aldehyde dehydrogenase from bacteria. In some embodiments, the NAD+ dependent aldehyde dehydrogenase is aldA (NP_415933) from E. coli. In another embodiment, the aldehyde dehydrogenase is a cytosolic NADP+ dependent aldehyde dehydrogenase from yeast. In some embodiments, the NADP+ dependent aldehyde dehydrogenase is ALD6 (YPL061W) from S. cerevisiae. In another embodiment, the aldehyde dehydrogenase is a cytosolic NADP+ dependent aldehyde dehydrogenase from bacteria. In some embodiments, the NADP+ dependent aldehyde dehydrogenase is aldB (NP_418045) from E. coli.
In one embodiment, overexpression of an enzyme to increase intracellular levels of a coenzyme comprises coupling supplementation of a co-substrate and overexpression of the enzyme. In one embodiment, the overexpression of an enzyme coupled with supplementation of a co-substrate of that enzyme increase flux through a biochemical pathway. In one embodiment, an NAD+ or NADP+ dependent alcohol dehydrogenase is expressed with a co-substrate. In certain embodiments, an alcohol dehydrogenase is expressed with an isopropanol co-substrate. In one embodiment, an NAD+ or NADP+ dependent glucose dehydrogenase is expressed with a co-substrate. In certain embodiments, a glucose dehydrogenase is expressed with a glucose co-substrate.
In one embodiment, the expression of a transhydrogenase is increased to interconvert NADH and NADPH. In some embodiments, the transhydrogenase is a pyridine nucleotide transhydrogenase. In some embodiments, the pyridine nucleotide transhydrogenase is from bacteria. In certain embodiments, the pyridine nucleotide transhydrogenase is pntAB (beta subunit: NP_416119; alpha subunit: NP_416120) from E. coli. In some embodiments, the pyridine nucleotide transhydrogenase is from human. In certain embodiments, the pyridine nucleotide transhydrogenase is NNT (NP_036475) from Homo sapiens. In certain embodiments, the pyridine nucleotide transhydrogenase is from Solanum tuberosum. In certain embodiments, the pyridine nucleotide transhydrogenase is from Spinacea oleracea.
In some embodiments, it may be useful to increase the expression of endogenous or exogenous proteins to induce endoplasmic reticulum (ER) membrane proliferation. In some embodiments, the induction of endoplasmic reticulum membrane proliferation can improve production of fatty alcohols, aldehydes, or acetates. In one embodiment, the expression of an inactivated HMG-CoA reductase (hydroxymethylglutaryl-CoA reductase) containing one or more ER facing loops is increased. In certain embodiments, the one or more loops is between transmembrane domains 6 and 7 of an inactivated HMG-CoA reductase. In some embodiments, the inactivated HMG-CoA reductase comprises an inactivated protein or chimera which codes for the first 500 amino acids or a subsequence of the first 500 amino acids of Yarrowia lipolytica YALI0E04807p. In other embodiments, the inactivated HMG-CoA reductase comprises an inactivated protein or chimera which codes for the first 522 amino acids or a subsequence of the first 522 amino acids of HMG1 from Saccharomyces cerevisiae (NP_013636.1). In other embodiments, the inactivated HMG-CoA reductase comprises an inactivated protein or chimera which codes for the first 522 amino acids or a subsequence of the first 522 amino acids of HMG2 from Saccharomyces cerevisiae (NP_013555.1). In some embodiments, the expression of one or more regulatory proteins is increased to improve production of fatty alcohols, aldehydes, or acetates. In certain embodiments, the regulatory protein comprises HAC1 transcription factor from Saccharomyces cerevisiae (NP_116622.1). In certain embodiments, the regulatory protein comprises HAC1 transcription factor from Yarrowia lipolytica (YALI0B12716p).
Increased synthesis or accumulation can be accomplished by, for example, overexpression of nucleic acids encoding one or more of the above-described a fatty alcohol pathway enzymes. Overexpression of a fatty alcohol pathway enzyme or enzymes can occur, for example, through increased expression of an endogenous gene or genes, or through the expression, or increased expression, of an exogenous gene or genes. Therefore, naturally occurring organisms can be readily modified to generate non-natural, fatty alcohol producing microorganisms through overexpression of one or more nucleic acid molecules encoding a fatty alcohol biosynthetic pathway enzyme. In addition, a non-naturally occurring organism can be generated by mutagenesis of an endogenous gene that results in an increase in activity of an enzyme in the fatty alcohol biosynthetic pathways.
Equipped with the present disclosure, the skilled artisan will be able to readily construct the recombinant microorganisms described herein, as the recombinant microorganisms of the disclosure can be constructed using methods well known in the art as exemplified above to exogenously express at least one nucleic acid encoding a fatty alcohol pathway enzyme in sufficient amounts to produce a fatty alcohol.
Methods for constructing and testing the expression levels of a non-naturally occurring fatty alcohol-producing host can be performed, for example, by recombinant and detection methods well known in the art. Such methods can be found described in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); Ausubo et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1999).
A variety of mechanisms known in the art can be used to express, or overexpress, exogenous or endogenous genes. For example, an expression vector or vectors can be constructed to harbor one or more fatty alcohol biosynthetic pathway enzyme encoding nucleic acids as exemplified herein operably linked to expression control sequences functional in the host organism. Expression vectors applicable for use in the microbial host organisms of the invention include, for example, plasmids, phage vectors, viral vectors, episomes and artificial chromosomes, including vectors and selection sequences or markers operable for stable integration into a host chromosome.
Selectable marker genes also can be included that, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media. In some embodiments, the present disclosure teaches the use of the bla (bacterial ampR resistance marker). In some embodiments, the present disclosure teaches use of the URA3 marker. In some embodiments, the present disclosure teaches microorganisms comprising the SUC2 gene to permit fermentation in sucrose media.
Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like which are well known in the art. When two or more exogenous encoding nucleic acids are to be co-expressed, both nucleic acids can be inserted, for example, into a single expression vector or in separate expression vectors. For single vector expression, the encoding nucleic acids can be operationally linked to one common expression control sequence or linked to different expression control sequences, such as one inducible promoter and one constitutive promoter. The transformation of exogenous nucleic acid sequences involved in a metabolic or synthetic pathway can be confirmed using methods well known in the art.
Expression control sequences are known in the art and include, for example, promoters, enhancers, polyadenylation signals, transcription terminators, internal ribosome entry sites (IRES), and the like, that provide for the expression of the polynucleotide sequence in a host cell. Expression control sequences interact specifically with cellular proteins involved in transcription (Maniatis et al., Science, 236: 1237-1245 (1987)). Exemplary expression control sequences are described in, for example, Goeddel, Gene Expression Technology: Methods in Enzymology, Vol. 185, Academic Press, San Diego, Calif. (1990).
In various embodiments, an expression control sequence may be operably linked to a polynucleotide sequence. By “operably linked” is meant that a polynucleotide sequence and an expression control sequence(s) are connected in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the expression control sequence(s). Operably linked promoters are located upstream of the selected polynucleotide sequence in terms of the direction of transcription and translation. Operably linked enhancers can be located upstream, within, or downstream of the selected polynucleotide.
In some embodiments, the recombinant microorganism is manipulated to delete, disrupt, mutate, and/or reduce the activity of one or more endogenous enzymes that catalyzes a reaction in a pathway that competes with the biosynthesis pathway for the production of a mono- or poly-unsaturated C6-C24 fatty alcohol, aldehyde, or acetate.
In some embodiments, the recombinant microorganism is manipulated to delete, disrupt, mutate, and/or reduce the activity of one or more endogenous enzymes that catalyzes the conversion of a fatty acid into a w-hydroxyfatty acid. In some such embodiments, the enzymes that catalyze the conversion of a fatty acid into a w-hydroxyfatty acid are selected from the group consisting of XP_504406, XP_504857, XP_504311, XP_500855, XP_500856, XP_500402, XP_500097, XP_501748, XP_500560, XP_501148, XP_501667, XP_500273, BAA02041, CAA39366, CAA39367, BAA02210, BAA02211, BAA02212, BAA02213, BAA02214, AAO73952, AAO73953, AAO73954, AAO73955, AAO73956, AAO73958, AAO73959, AAO73960, AAO73961, AAO73957, XP_002546278, BAM49649, AAB80867, AAB17462, ADL27534, AAU24352, AAA87602, CAA34612, ABM17701, AAA25760, CAB51047, AAC82967, WP 011027348, or homologs thereof.
In some embodiments, the recombinant microorganism is manipulated to delete, disrupt, mutate, and/or reduce the activity of one or more endogenous cytochrome P450 monooxygenases selected from the group consisting of Y. lipolytica YALI0E25982g (ALK1), Y. lipolytica YALI0F01320g (ALK2), Y. lipolytica YALI0E23474g (ALK3), Y. lipolytica YALI0B13816g (ALK4), Y. lipolytica YALI0B13838g (ALK5), Y. lipolytica YALI0B01848g (ALK6), Y. lipolytica YALI0A15488g (ALK7), Y. lipolytica YALI0C12122g (ALK8), Y. lipolytica YALI0B06248g (ALK9), Y. lipolytica YALI0B20702g (ALK10), Y. lipolytica YALI0C10054g (ALK11) and Y. lipolytica YALI0A20130g (ALK12).
In other embodiments, the recombinant microorganism is manipulated to delete, disrupt, mutate, and/or reduce the activity of one or more endogenous enzymes that catalyzes the conversion of a fatty acyl-CoA into α,β-enoyl-CoA. In some such embodiments, the enzymes that catalyze the conversion of a fatty acyl-CoA into α,β-enoyl-CoA are selected from the group consisting of CAA04659, CAA04660, CAA04661, CAA04662, CAA04663, CAG79214, AAA34322, AAA34361, AAA34363, CAA29901, BAA04761, AAA34891, AAB08643, CAB15271, BAN55749, CAC44516, ADK16968, AEI37634, WP_000973047, WP_025433422, WP_035184107, WP_026484842, CEL80920, WP_026818657, WP_005293707, WP_005883960, or homologs thereof.
In some embodiments, one or more genes of the microbial host encoding acyl-CoA oxidases are deleted or down-regulated to eliminate or reduce the truncation of desired fatty acyl-CoAs beyond a desired chain-length. Such deletion or down-regulation targets include but are not limited to Y. lipolytica POX1(YALI0E32835g), Y. lipolytica POX2(YALI0F10857g), Y. lipolytica POX3(YALI0D24750g), Y. lipolytica POX4(YALI0E27654g), Y. lipolytica POX5(YALI0C23859g), Y. lipolytica POX6(YALI0E06567g); S. cerevisiae POX1(YGL205W); Candida POX2 (CaO19.1655, CaO19.9224, CTRG_02374, M18259), Candida POX4 (CaO19.1652, CaO19.9221, CTRG_02377, M12160), and Candida POX5 (CaO19.5723, CaO19.13146, CTRG_02721, M12161).
In some embodiments, the recombinant microorganism is manipulated to delete, disrupt, mutate, and/or reduce the activity of one or more proteins involved in peroxisome biogenesis. In such embodiments, the one or more proteins involved in peroxisome biogenesis are selected from the group consisting of XP_505754, XP_501986, XP_501311, XP_504845, XP_503326, XP_504029, XP_002549868, XP_002547156, XP_002545227, XP_002547350, XP_002546990, EIW11539, EIW08094, EIW11472, EIW09743, EIW0828, or homologs thereof.
In some embodiments, the recombinant microorganism is manipulated to delete, disrupt, mutate, and/or reduce the activity of one or more endogenous enzymes that catalyzes a reaction in a pathway that competes with the biosynthesis pathway for one or more unsaturated fatty acyl-CoA intermediates. In one embodiment, the one or more endogenous enzymes comprise one or more diacylglycerol acyltransferases. In the context of a recombinant yeast microorganism, the recombinant yeast microorganism is engineered to delete, disrupt, mutate, and/or reduce the activity of one or more diacylglycerol acyltransferases selected from the group consisting of YALI0E32769g, YALI0D07986g and CTRG_06209, or homolog thereof. In another embodiment, the one or more endogenous enzymes comprise one or more glycerolphospholipid acyltransferases. In the context of a recombinant yeast microorganism, the recombinant yeast microorganism is engineered to delete, disrupt, mutate, and/or reduce the activity of one or more glycerolphospholipid acyltransferases selected from the group consisting of YALI0E16797g and CTG_04390, or homolog thereof. In another embodiment, the one or more endogenous enzymes comprise one or more acyl-CoA/sterol acyltransferases. In the context of a recombinant yeast microorganism, the recombinant yeast microorganism is engineered to delete, disrupt, mutate, and/or reduce the activity of one or more acyl-CoA/sterol acyltransferases selected from the group consisting of YALI0F06578g, CTRG_01764 and CTRG_01765, or homolog thereof.
In some embodiments, one or more genes of the microbial host encoding glycerol-3-phosphate acyl transferases (GPATs), lysophosphatidic acid acyltransferases (LPAATs), glycerolphospholipid acyltransferase (GPLATs) and/or diacylglycerol acyltransferases (DGATs) are deleted or downregulated, and replaced with one or more GPATs, LPAATs, GPLATs, or DGATs which prefer to store short-chain fatty acyl-CoAs. Such deletion or downregulation targets include but are not limited to Y. lipolytica YALI0000209g, Y. lipolytica YALI0E18964g, Y. lipolytica YALI0F19514g, Y. lipolytica Y. lipolytica YALI0C14014g, Y. lipolytica YALI0E16797g, Y. lipolytica YALI0E32769g, Y. lipolytica YALI0D07986g, S. cerevisiae YBL011w, S. cerevisiae YDL052c, S. cerevisiae YOR175C, S. cerevisiae YPR139C, S. cerevisiae YNR008w, S. cerevisiae YOR245c, Candida I503_02577, Candida CTRG_02630, Candida CaO19.250, Candida CaO19.7881, Candida CTRG_02437, Candida CaO19.1881, Candida CaO19.9437, Candida CTRG_01687, Candida CaO19.1043, Candida CaO19.8645, Candida CTRG_04750, Candida CaO19.13439, Candida CTRG_04390, Candida CaO19.6941, Candida CaO19.14203, and Candida CTRG_06209.
In some preferred embodiments, one or more genes of the microbial host encoding acylglycerol lipases (mono-, di-, or triacylglycerol lipases) and sterol ester esterases are deleted or downregulated and replaced with one or more acylglycerol lipases which prefer long chain acylglycerol substrates. In some embodiments, the one or more endogenous acylglycerol lipase and/or sterol ester esterase enzymes being deleted or downregulated are selected from Y. lipolytica YALI0E32035g, Y. lipolytica YALI0D17534g, Y. lipolytica YALI0F10010g, Y. lipolytica YALI0C14520g, Y. lipolytica YALI0E00528g, S. cerevisiae YKL140w, S. cerevisiae YMR313c, S. cerevisiae YKR089c, S. cerevisiae YOR081c, S. cerevisiae YKL094W, S. cerevisiae YLL012W, S. cerevisiae YLR020C, Candida CaO19.2050, Candida CaO19.9598, Candida CTRG_01138, Candida WSQ_03398, Candida CTRG_00057, Candida CaO19.5426, Candida CaO19.12881, Candida CTRG_06185, Candida CaO19.4864, Candida CaO19.12328, Candida CTRG_03360, Candida CaO19.6501, Candida CaO19.13854, Candida CTRG_05049, Candida CaO19.1887, Candida CaO19.9443, Candida CTRG_01683, and Candida CTRG_04630.
In another embodiment, the recombinant microorganism is manipulated to delete, disrupt, mutate, and/or reduce the activity of one or more endogenous enzymes that catalyzes a reaction in a pathway that oxidizes fatty aldehyde intermediates. In one embodiment, the one or more endogenous enzymes comprise one or more fatty aldehyde dehydrogenases. In the context of a recombinant yeast microorganism, the recombinant yeast microorganism is engineered to delete, disrupt, mutate, and/or reduce the activity of one or more fatty aldehyde dehydrogenases selected from the group consisting of YALI0A17875g, YALI0E15400g, YALI0B01298g, YALI0F23793g, CTRG_05010 and CTRG_04471, or homolog thereof.
In another embodiment, the recombinant microorganism is manipulated to delete, disrupt, mutate, and/or reduce the activity of one or more endogenous enzymes that catalyzes a reaction in a pathway that consumes fatty acetate products. In one embodiment, the one or more endogenous enzymes comprise one or more sterol esterases. In the context of a recombinant yeast microorganism, the recombinant yeast microorganism is engineered to delete, disrupt, mutate, and/or reduce the activity of one or more sterol esterases selected from the group consisting of YALI0E32035g, YALI0E00528g, CTRG_01138, CTRG_01683 and CTRG_04630, or homolog thereof. In another embodiment, the one or more endogenous enzymes comprise one or more triacylglycerol lipases. In the context of a recombinant yeast microorganism, the recombinant yeast microorganism is engineered to delete, disrupt, mutate, and/or reduce the activity of one or more triacylglycerol lipases selected from the group consisting of YALI0D17534g, YALI0F10010g, CTRG_00057 and CTRG_06185, or homolog thereof. In another embodiment, the one or more endogenous enzymes comprise one or more monoacylglycerol lipases. In the context of a recombinant yeast microorganism, the recombinant yeast microorganism is engineered to delete, disrupt, mutate, and/or reduce the activity of one or more monoacylglycerol lipases selected from the group consisting of YALI0C14520g, CTRG_03360 and CTRG_05049, or homolog thereof. In another embodiment, the one or more endogenous enzymes comprise one or more extracellular lipases. In the context of a recombinant yeast microorganism, the recombinant yeast microorganism is engineered to delete, disrupt, mutate, and/or reduce the activity of one or more extracellular lipases selected from the group consisting of YALI0A20350g, YALI0D19184g, YALI0B09361g, CTRG_05930, CTRG_04188, CTRG_02799, CTRG_03052 and CTRG_03885, or homolog thereof.
In some embodiments, the recombinant microorganism is manipulated to delete, disrupt, mutate, and/or reduce the activity of one or more endogenous enzymes that (1) break down fatty acids in the course of beta-oxidation and/or (2) oxidize w-hydroxy fatty acids to fatty acid aldehyde or to dicarboxylic acid in the course of w-oxidation. In some embodiments, the recombinant microorganism comprises a deletion, disruption, mutation, and/or reduction in the activity of: (i) one or more endogenous acyl-CoA oxidase selected from the group consisting of YALI0E32835g (POX1), YALI0F10857g (POX2), YALI0D24750g (POX3), YALI0E27654g (POX4), YALI0C23859g (POX5), YALI0E06567g (POX6); (ii) one or more endogenous (fatty) alcohol dehydrogenase selected from the group consisting of YALI0F09603g (FADH), YALI0D25630g (ADH1), YALI0E17787g (ADH2), YALI0A16379g (ADH3), YALI0E15818g (ADH4), YALI0D02167g (ADH5), YALI0A15147g (ADH6), YALI0E07766g (ADH7); and (iii) an endogenous (fatty) alcohol oxidase YALI0B14014g (FAO1).
In some embodiments, the Y. lipolytica microorganism into which biosynthesis pathways for the production of C6-C24 fatty alcohol, fatty aldehyde and/or fatty acetate are introduced is H222 ΔP ΔA ΔF ΔURA3. AP denotes deletion of the acyl-CoA oxidase genes (POX 1-6) in Y. lipolytica. ΔA denotes deletion of the (fatty) alcohol dehydrogenase genes (FADH, ADH 1-7) in Y. lipolytica. ΔF denotes deletion of the (fatty) alcohol oxidase gene (FAO1) in Y. lipolytica. ΔURA3 denotes deletion of the URA3 gene in Y. lipolytica, rendering the yeast a uracil auxotroph. In some embodiments, the Y. lipolytica microorganism into which biosynthesis pathways for the production of C6-C24 fatty alcohol, fatty aldehyde and/or fatty acetate are introduced is H222 ΔP ΔA ΔF. In some embodiments, the Y. lipolytica microorganism into which biosynthesis pathways for the production of C6-C24 fatty alcohol, fatty aldehyde and/or fatty acetate are introduced is MATA ura3-302::SUC2 Δpox1 Δpox2 Δpox3 Δpox4 Δpox5 Δpox6 Δfadh Δadh1 Δadh2 Δadh3 Δadh4 Δadh5 Δadh6 Δadh7 Δfao1::URA3.
A wild type isolate of the yeast Y. lipolytica, preferably of the strain H222, can be used as the starting strain for the construction of strains according to the disclosure. The strain H222 was deposited on 29 Apr. 2013 at the DSMZ (Deutsche Sammlung fur Mikroorganismen and Zellkulturen GmbH, D-38142 Braunschweig) under the number DSM 27185 according to the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. A selection marker is required for the use of a strain for further genetic processing. This selection marker can be introduced into the strain in a manner known per se, e.g. in the form of the uracil auxotroph. Alternatively, already known uracil auxotrophic strains can be used, preferably the strain H222-S4 (Mauersberger S, Wang H J, Gaillard in C, Barth G & Nicaud J M (2001) J Bacterial 183: 5102-5109). The respective deletion cassette (e.g. POX 1-6, FADH, ADH 1-7, FAO1) is obtained by PCR or restriction and transformed into Y. lipolytica H222-S4, which can be produced from Y. lipolytica H222 (Mauers-berger et al. (2001)), according to Barth and Gaillardin (Barth G & Gaillardin C (1996) Yarrowia lipolytica. Springer-Verlag, Berlin, Heidelberg, New York). The creation of H222 ΔP ΔA ΔF ΔURA3 is described in WO 2015/086684, which is herein incorporated by reference in its entirety. Y. lipolytica strain H222 ΔP ΔA ΔF ΔURA3 is used as the starting microorganism for introduction of desaturases and reductases in the present disclosure (see, for example, Examples 7, 9 and 10).
In another embodiment, the recombinant microorganism is manipulated to delete, disrupt, mutate, and/or reduce the activity of one or more endogenous reductase or desaturase enzymes that interferes with the unsaturated C6-C24 fatty alcohol, aldehyde, or acetate, i.e., catalyzes the conversion of a pathway substrate or product into an unwanted by-product.
Chemical Conversion of Product from Microorganism Synthesis
The present disclosure describes chemical conversions that can be used to convert a product synthesized by recombinant microorganism into a down-stream product.
In some embodiments, an unsaturated fatty alcohol, aldehyde, acetate, or carboxylic acid produced by a microorganism can undergo subsequent chemical conversion to produce a pheromone, fragrance, flavor, polymer, or polymer intermediate. Non-limiting examples of chemical transformations include esterification, metathesis, and polymerization.
Unsaturated fatty carboxylic acids can be esterified by methods known in the art. For example, Fischer esterification can be used to covert a fatty carboxylic acid to a corresponding fatty ester. See, e.g., Komura, K. et al., Synthesis 2008. 3407-3410.
Elongation of the carbon chain can be performed by known methods to covert an unsaturated fatty alcohol into an elongated derivative thereof. Olefin metastasis catalysts can be performed to increase the number of carbons on the fatty carbon chain and impart Z or E stereochemistry on the corresponding unsaturated product.
In some embodiments, the metathesis catalyst is a tungsten metathesis catalyst, a molybdenum metathesis catalyst, or a ruthenium metathesis catalyst. In certain embodiments, the metathesis catalyst is a tungsten catalyst or a molybdenum catalyst. The catalysts employed in the present invention generally employ metals which can mediate a particular desired chemical reaction. In general, any transition metal (e.g., having d electrons) can be used to form the catalyst, e.g., a metal selected from one of Groups 3-12 of the periodic table or from the lanthanide series. In some embodiments, the metal is selected from Groups 3-8, or, in some cases, from Groups 4-7. In some embodiments, the metal is selected from Group 6. The term “Group 6” refers to the transition metal group comprising chromium, molybdenum, and tungsten. Additionally, the present invention may also include the formation of heterogeneous catalysts containing forms of these elements (e.g., by immobilizing a metal complex on an insoluble substrate, for example, silica).
In general, any metathesis catalyst stable under the reaction conditions and nonreactive with functional groups on the fatty substrate (e.g., alcohol, ester, carboxylic acid, aldehyde, or acetate) can be used with the present disclosure. Such catalysts are, for example, those described by Grubbs (Grubbs, R. H., “Synthesis of large and small molecules using olefin metathesis catalysts.” PMSE Prepr., 2012), herein incorporated by reference in its entirety. Depending on the desired isomer of the olefin, as cis-selective metathesis catalyst may be used, for example one of those described by Shahane et al. (Shahane, S., et al. ChemCatChem, 2013. 5(12): p. 3436-3459), herein incorporated by reference in its entirety. Catalysts exhibiting cis-selectivity have been described previously (Khan, R. K., et al. J. Am. Chem. Soc., 2013. 135(28): p. 10258-61; Hartung, J. et al. J. Am. Chem. Soc., 2013. 135(28): p. 10183-5.; Rosebrugh, L. E., et al. J. Am. Chem. Soc., 2013. 135(4): p. 1276-9.; Marx, V. M., et al. J. Am. Chem. Soc., 2013. 135(1): p. 94-7.; Herbert, M. B., et al. Angew. Chem. Int. Ed. Engl., 2013. 52(1): p. 310-4; Keitz, B. K., et al. J. Am. Chem. Soc., 2012. 134(4): p. 2040-3; Keitz, B. K., et al. J. Am. Chem. Soc., 2012. 134(1): p. 693-9.; Endo, K. et al. J. Am. Chem. Soc., 2011. 133(22): p. 8525-7).
Additional Z-selective catalysts are described in (Cannon and Grubbs 2013; Bronner et al. 2014; Hartung et al. 2014; Pribisko et al. 2014; Quigley and Grubbs 2014) and are herein incorporated by reference in their entirety. Due to their excellent stability and functional group tolerance, in some embodiments metathesis catalysts include, but are not limited to, neutral ruthenium or osmium metal carbene complexes that possess metal centers that are formally in the +2 oxidation state, have an electron count of 16, are penta-coordinated, and are of the general formula LL′AA′M=CRbRc or LL′AA′M=(C=)nCRbRc (Pederson and Grubbs 2002); wherein
M is ruthenium or osmium;
L and L′ are each independently any neutral electron donor ligand and selected from phosphine, sulfonated phosphine, phosphite, phosphinite, phosphonite, arsine, stibnite, ether, amine, amide, imine, sulfoxide, carboxyl, nitrosyl, pyridine, thioether, or heterocyclic carbenes; and
A and A′ are anionic ligands independently selected from halogen, hydrogen, C1-C20 alkyl, aryl, C1-C20 alkoxide, aryloxide, C2-C20 alkoxycarbonyl, arylcarboxylate, C1-C20 carboxylate, arylsulfonyl, C1-C20 alkylsulfonyl, C1-C20 alkylsulfinyl; each ligand optionally being substituted with C1-C5 alkyl, halogen, C1-C5 alkoxy; or with a phenyl group that is optionally substituted with halogen, C1-C5 alkyl, or C1-C5 alkoxy; and A and A′ together may optionally comprise a bidentate ligand; and
Rb and Rc are independently selected from hydrogen, C1-C20 alkyl, aryl, C1-C20 carboxylate, C1-C20 alkoxy, aryloxy, C1-C20 alkoxycarbonyl, C1-C20 alkylthio, C1-C20 alkylsulfonyl and C1-C20 alkylsulfinyl, each of Rb and Rc optionally substituted with C1-C5 alkyl, halogen, C1-C5 alkoxy or with a phenyl group that is optionally substituted with halogen, C1-C5 alkyl, or C1-C5 alkoxy.
Other metathesis catalysts such as “well defined catalysts” can also be used. Such catalysts include, but are not limited to, Schrock's molybdenum metathesis catalyst, 2,6-diisopropylphenylimido neophylidenemolybdenum (VI) bis(hexafluoro-t-butoxide), described by Grubbs et al. (Tetrahedron 1998, 54: 4413-4450) and Basset's tungsten metathesis catalyst described by Couturier, J. L. et al. (Angew. Chem. Int. Ed. Engl. 1992, 31: 628).
Catalysts useful in the methods of the disclosure also include those described by U.S. Pat. No. 9,776,179, Peryshkov, et al. J. Am. Chem. Soc. 2011, 133: 20754-20757; Wang, et al. Angewandte Chemie, 2013, 52: 1939-1943; Yu, et al. J. Am. Chem. Soc., 2012, 134: 2788-2799; Halford. Chem. Eng. News, 2011, 89 (45): 11; Yu, et al. Nature, 2011, 479: 88-93; Lee. Nature, 2011, 471: 452-453; Meek, et al. Nature, 2011: 471, 461-466; Flook, et al. J. Am. Chem. Soc. 2011, 133: 1784-1786; Zhao, et al. Org Lett, 2011, 13(4): 784-787; Ondi, et al. “High activity, stabilized formulations, efficient synthesis and industrial use of Mo- and W-based metathesis catalysts” XiMo Technology Updates, 2015: http://www.ximo-inc.com/files/ximo/uploads/download/Summary_3.11.15.pdf; Schrock, et al. Macromolecules, 2010: 43, 7515-7522; Peryshkov, et al. Organometallics 2013: 32, 5256-5259; Gerber, et al. Organometallics 2013: 32, 5573-5580; Marinescu, et al. Organometallics 2012: 31, 6336-6343; Wang, et al. Angew. Chem. Int. Ed. 2013: 52, 1939-1943; Wang, et al. Chem. Eur. 1 2013: 19, 2726-2740; and Townsend et al. J. Am. Chem. Soc. 2012: 134, 11334-11337.
Catalysts useful in the methods of the disclosure also include those described in International Pub. No. WO 2014/155185; International Pub. No. WO 2014/172534; U.S. Pat. Appl. Pub. No. 2014/0330018; International Pub. No. WO 2015/003815; and International Pub. No. WO 2015/003814.
Catalysts useful in the methods of the disclosure also include those described in U.S. Pat. Nos. 4,231,947; 4,245,131; 4,427,595; 4,681,956; 4,727,215; International Pub. No. WO 1991/009825; U.S. Pat. Nos. 5,087,710; 5,142,073; 5,146,033; International Pub. No. WO 1992/019631; U.S. Pat. Nos. 6,121,473; 6,346,652; 8,987,531; U.S. Pat. Appl. Pub. No. 2008/0119678; International Pub. No. WO 2008/066754; International Pub. No. WO 2009/094201; U.S. Pat. Appl. Pub. No. 2011/0015430; U.S. Pat. Appl. Pub. No. 2011/0065915; U.S. Pat. Appl. Pub. No. 2011/0077421; International Pub. No. WO 2011/040963; International Pub. No. WO 2011/097642; U.S. Pat. Appl. Pub. No. 2011/0237815; U.S. Pat. Appl. Pub. No. 2012/0302710; International Pub. No. WO 2012/167171; U.S. Pat. Appl. Pub. No. 2012/0323000; U.S. Pat. Appl. Pub. No. 2013/0116434; International Pub. No. WO 2013/070725; U.S. Pat. Appl. Pub. No. 2013/0274482; U.S. Pat. Appl. Pub. No. 2013/0281706; International Pub. No. WO 2014/139679; International Pub. No. WO 2014/169014; U.S. Pat. Appl. Pub. No. 2014/0330018; and U.S. Pat. Appl. Pub. No. 2014/0378637.
Catalysts useful in the methods of the disclosure also include those described in International Pub. No. WO 2007/075427; U.S. Pat. Appl. Pub. No. 2007/0282148; International Pub. No. WO 2009/126831; International Pub. No. WO 2011/069134; U.S. Pat. Appl. Pub. No. 2012/0123133; U.S. Pat. Appl. Pub. No. 2013/0261312; U.S. Pat. Appl. Pub. No. 2013/0296511; International Pub. No. WO 2014/134333; and U.S. Pat. Appl. Pub. No. 2015/0018557.
Catalysts useful in the methods of the disclosure also include those described in U.S. Pat. Appl. Pub. No. 2008/0009598; U.S. Pat. Appl. Pub. No. 2008/0207911; U.S. Pat. Appl. Pub. No. 2008/0275247; U.S. Pat. Appl. Pub. No. 2011/0040099; U.S. Pat. Appl. Pub. No. 2011/0282068; and U.S. Pat. Appl. Pub. No. 2015/0038723.
Catalysts useful in the methods of the disclosure include those described in International Pub. No. WO 2007/140954; U.S. Pat. Appl. Pub. No. 2008/0221345; International Pub. No. WO 2010/037550; U.S. Pat. Appl. Pub. No. 2010/0087644; U.S. Pat. Appl. Pub. No. 2010/0113795; U.S. Pat. Appl. Pub. No. 2010/0174068; International Pub. No. WO 2011/091980; International Pub. No. WO 2012/168183; U.S. Pat. Appl. Pub. No. 2013/0079515; U.S. Pat. Appl. Pub. No. 2013/0144060; U.S. Pat. Appl. Pub. No. 2013/0211096; International Pub. No. WO 2013/135776; International Pub. No. WO 2014/001291; International Pub. No. WO 2014/067767; U.S. Pat. Appl. Pub. No. 2014/0171607; and U.S. Pat. Appl. Pub. No. 2015/0045558.
The catalyst is typically provided in the reaction mixture in a sub-stoichiometric amount (e.g., catalytic amount). In certain embodiments, that amount is in the range of about 0.001 to about 50 mol % with respect to the limiting reagent of the chemical reaction, depending upon which reagent is in stoichiometric excess. In some embodiments, the catalyst is present in less than or equal to about 40 mol % relative to the limiting reagent. In some embodiments, the catalyst is present in less than or equal to about 30 mol % relative to the limiting reagent. In some embodiments, the catalyst is present in less than about 20 mol %, less than about 10 mol %, less than about 5 mol %, less than about 2.5 mol %, less than about 1 mol %, less than about 0.5 mol %, less than about 0.1 mol %, less than about 0.015 mol %, less than about 0.01 mol %, less than about 0.0015 mol %, or less, relative to the limiting reagent. In some embodiments, the catalyst is present in the range of about 2.5 mol % to about 5 mol %, relative to the limiting reagent. In some embodiments, the reaction mixture contains about 0.5 mol % catalyst. In the case where the molecular formula of the catalyst complex includes more than one metal, the amount of the catalyst complex used in the reaction may be adjusted accordingly.
In some cases, the methods described herein can be performed in the absence of solvent (e.g., neat). In some cases, the methods can include the use of one or more solvents. Examples of solvents that may be suitable for use in the disclosure include, but are not limited to, benzene, p-cresol, toluene, xylene, diethyl ether, glycol, diethyl ether, petroleum ether, hexane, cyclohexane, pentane, methylene chloride, chloroform, carbon tetrachloride, dioxane, tetrahydrofuran (THF), dimethyl sulfoxide, dimethylformamide, hexamethyl-phosphoric triamide, ethyl acetate, pyridine, triethylamine, picoline, and the like, as well as mixtures thereof. In some embodiments, the solvent is selected from benzene, toluene, pentane, methylene chloride, and THF. In certain embodiments, the solvent is benzene.
In some embodiments, the method is performed under reduced pressure. This may be advantageous in cases where a volatile byproduct, such as ethylene, may be produced during the course of the metathesis reaction. For example, removal of the ethylene byproduct from the reaction vessel may advantageously shift the equilibrium of the metathesis reaction towards formation of the desired product. In some embodiments, the method is performed at a pressure of about less than 760 torr. In some embodiments, the method is performed at a pressure of about less than 700 torr. In some embodiments, the method is performed at a pressure of about less than 650 torr. In some embodiments, the method is performed at a pressure of about less than 600 torr. In some embodiments, the method is performed at a pressure of about less than 550 torr. In some embodiments, the method is performed at a pressure of about less than 500 torr. In some embodiments, the method is performed at a pressure of about less than 450 torr. In some embodiments, the method is performed at a pressure of about less than 400 torr. In some embodiments, the method is performed at a pressure of about less than 350 torr. In some embodiments, the method is performed at a pressure of about less than 300 torr. In some embodiments, the method is performed at a pressure of about less than 250 torr. In some embodiments, the method is performed at a pressure of about less than 200 torr. In some embodiments, the method is performed at a pressure of about less than 150 torr. In some embodiments, the method is performed at a pressure of about less than 100 torr. In some embodiments, the method is performed at a pressure of about less than 90 torr. In some embodiments, the method is performed at a pressure of about less than 80 torr. In some embodiments, the method is performed at a pressure of about less than 70 torr. In some embodiments, the method is performed at a pressure of about less than 60 torr. In some embodiments, the method is performed at a pressure of about less than 50 torr. In some embodiments, the method is performed at a pressure of about less than 40 torr. In some embodiments, the method is performed at a pressure of about less than 30 torr. In some embodiments, the method is performed at a pressure of about less than 20 torr. In some embodiments, the method is performed at a pressure of about 20 torr. In some embodiments, the method is performed at a pressure of about 10 torr. In some embodiments, the method is performed at a pressure of about 10 torr. In some embodiments, the method is performed at a pressure of about 1 torr. In some embodiments, the method is performed at a pressure of less than about 1 torr.
In some embodiments, the two metathesis reactants are present in equimolar amounts. In some embodiments, the two metathesis reactants are not present in equimolar amounts. In certain embodiments, the two reactants are present in a molar ratio of about 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, or 1:20. In certain embodiments, the two reactants are present in a molar ratio of about 10:1. In certain embodiments, the two reactants are present in a molar ratio of about 7:1. In certain embodiments, the two reactants are present in a molar ratio of about 5:1. In certain embodiments, the two reactants are present in a molar ratio of about 2:1. In certain embodiments, the two reactants are present in a molar ratio of about 1:10. In certain embodiments, the two reactants are present in a molar ratio of about 1:7. In certain embodiments, the two reactants are present in a molar ratio of about 1:5. In certain embodiments, the two reactants are present in a molar ratio of about 1:2.
In general, the reactions with many of the metathesis catalysts disclosed herein provide yields better than 15%, better than 50%, better than 75%, or better than 90%. In addition, the reactants and products are chosen to provide at least a 5° C. difference, a greater than 20° C. difference, or a greater than 40° C. difference in boiling points. Additionally, the use of metathesis catalysts allows for much faster product formation than byproduct, it is desirable to run these reactions as quickly as practical. In particular, the reactions are performed in less than about 24 hours, less than 12 hours, less than 8 hours, or less than 4 hours.
One of skill in the art will appreciate that the time, temperature and solvent can depend on each other, and that changing one can require changing the others to prepare the pyrethroid products and intermediates in the methods of the disclosure. The metathesis steps can proceed at a variety of temperatures and times. In general, reactions in the methods of the disclosure are conducted using reaction times of several minutes to several days. For example, reaction times of from about 12 hours to about 7 days can be used. In some embodiments, reaction times of 1-5 days can be used. In some embodiments, reaction times of from about 10 minutes to about 10 hours can be used. In general, reactions in the methods of the disclosure are conducted at a temperature of from about 0° C. to about 200° C. For example, reactions can be conducted at 15-100° C. In some embodiments, reaction can be conducted at 20-80° C. In some embodiments, reactions can be conducted at 100-150° C.
Unsaturated fatty esters can be reduced using a suitable reducing agent which selectively reduces the ester to the corresponding aldehyde or alcohol but does not reduce the double bond. An unsaturated fatty ester can be reduced to the corresponding unsaturated fatty aldehyde using di-isobutyl aluminum halide (DIBAL) or Vitride®. The unsaturated fatty aldehyde can be reduced to the corresponding fatty alcohol with, e.g., DIBAL or Vitride®. In some embodiments, the unsaturated fatty ester can be reduced to the corresponding fatty alcohol using AlH3 or 9-Borabicyclo(3.3.1)nonane (9-BBN). (See Galatis, P. Encyclopedia of Reagents for Organic Synthesis. 2001. New York: John Wiley & Sons; and Carey & Sunderburg. Organic Chemistry, Part B: Reactions and Synthesis, 5th edition. 2007. New York Springer Sciences.)
Pheromone Compositions and Uses Thereof
As described above, products made via the methods described herein are pheromones. Pheromones prepared according to the methods of the invention can be formulated for use as insect control compositions. The pheromone compositions can include a carrier, and/or be contained in a dispenser. The carrier can be, but is not limited to, an inert liquid or solid.
Examples of solid carriers include but are not limited to fillers such as kaolin, bentonite, dolomite, calcium carbonate, talc, powdered magnesia, Fuller's earth, wax, gypsum, diatomaceous earth, rubber, plastic, China clay, mineral earths such as silicas, silica gels, silicates, attaclay, limestone, chalk, loess, clay, dolomite, calcium sulfate, magnesium sulfate, magnesium oxide, ground synthetic materials, fertilizers such as ammonium sulfate, ammonium phosphate, ammonium nitrate, thiourea and urea, products of vegetable origin such as cereal meals, tree bark meal, wood meal and nutshell meal, cellulose powders, attapulgites, montmorillonites, mica, vermiculites, synthetic silicas and synthetic calcium silicates, or compositions of these.
Examples of liquid carriers include, but are not limited to, water; alcohols, such as ethanol, butanol or glycol, as well as their ethers or esters, such as methylglycol acetate; ketones, such as acetone, cyclohexanone, methylethyl ketone, methylisobutylketone, or isophorone; alkanes such as hexane, pentane, or heptanes; aromatic hydrocarbons, such as xylenes or alkyl naphthalenes; mineral or vegetable oils; aliphatic chlorinated hydrocarbons, such as trichloroethane or methylene chloride; aromatic chlorinated hydrocarbons, such as chlorobenzenes; water-soluble or strongly polar solvents such as dimethylformamide, dimethyl sulfoxide, or N-methylpyrrolidone; liquefied gases; waxes, such as beeswax, lanolin, shellac wax, carnauba wax, fruit wax (such as bayberry or sugar cane wax) candelilla wax, other waxes such as microcrystalline, ozocerite, ceresin, or montan; salts such as monoethanolamine salt, sodium sulfate, potassium sulfate, sodium chloride, potassium chloride, sodium acetate, ammonium hydrogen sulfate, ammonium chloride, ammonium acetate, ammonium formate, ammonium oxalate, ammonium carbonate, ammonium hydrogen carbonate, ammonium thiosulfate, ammonium hydrogen diphosphate, ammonium dihydrogen monophosphate, ammonium sodium hydrogen phosphate, ammonium thiocyanate, ammonium sulfamate or ammonium carbamate and mixtures thereof. Baits or feeding stimulants can also be added to the carrier.
Synergist
In some embodiments, the pheromone composition is combined with an active chemical agent such that a synergistic effect results. The synergistic effect obtained by the taught methods can be quantified according to Colby's formula (i.e. (E)=X+Y−(X*Y/100). See Colby, R. S., “Calculating Synergistic and Antagonistic Responses of Herbicide Combinations”, 1967 Weeds, vol. 15, pp. 20-22, incorporated herein by reference in its entirety. Thus, by “synergistic” is intended a component which, by virtue of its presence, increases the desired effect by more than an additive amount. The pheromone compositions and adjuvants of the present methods can synergistically increase the effectiveness of agricultural active compounds and also agricultural auxiliary compounds.
Thus, in some embodiments, a pheromone composition can be formulated with a synergist. The term, “synergist,” as used herein, refers to a substance that can be used with a pheromone for reducing the amount of the pheromone dose or enhancing the effectiveness of the pheromone for attracting at least one species of insect. The synergist may or may not be an independent attractant of an insect in the absence of a pheromone.
In some embodiments, the synergist is a volatile phytochemical that attracts at least one species of Lepidoptera. The term, “phytochemical,” as used herein, means a compound occurring naturally in a plant species. In a particular embodiment, the synergist is selected from the group comprising β-caryophyllene, iso-caryophyllene, α-humulene, inalool, Z3-hexenol/yl acetate, β-farnesene, benzaldehyde, phenylacetaldehyde, and combinations thereof.
The pheromone composition can contain the pheromone and the synergist in a mixed or otherwise combined form, or it may contain the pheromone and the synergist independently in a non-mixed form.
Insecticide
The pheromone composition can include one or more insecticides. In one embodiment, the insecticides are chemical insecticides known to one skilled in the art. Examples of the chemical insecticides include one or more of pyrethroid or organophosphorus insecticides, including but are not limited to, cyfluthrin, permethrin, cypermethrin, bifenthrin, fenvalerate, flucythrinate, azinphosmethyl, methyl parathion, buprofezin, pyriproxyfen, flonicamid, acetamiprid, dinotefuran, clothianidin, acephate, malathion, quinalphos, chloropyriphos, profenophos, bendiocarb, bifenthrin, chlorpyrifos, cyfluthrin, diazinon, pyrethrum, fenpropathrin, kinoprene, insecticidal soap or oil, neonicotinoids, diamides, avermectin and derivatives, spinosad and derivatives, azadirachtin, pyridalyl, and mixtures thereof.
In another embodiment, the insecticides are one or more biological insecticides known to one skilled in the art. Examples of the biological insecticides include, but are not limited to, azadirachtin (neem oil), toxins from natural pyrethrins, Bacillus thuringiensis and Beauveria bassiana, viruses (e.g., CYD-X™, CYD-X HP™, Germstar™, Madex HP™ and Spod-X™), peptides (Spear-T™, Spear-P™, and Spear-C™)
In another embodiment, the insecticides are insecticides that target the nerve and muscle. Examples include acetylcholinesterase (AChE) inhibitors, such as carbamates (e.g., methomyl and thiodicarb) and organophosphates (e.g., chlorpyrifos) GABA-gated chloride channel antagonists, such as cyclodiene organochlorines (e.g., endosulfan) and phenylpyrazoles (e.g., fipronil), sodium channel modulators, such as pyrethrins and pyrethroids (e.g., cypermethrin and λ-cyhalothrin), nicotinic acetylcholine receptor (nAChR) agonists, such as neonicotinoids (e.g., acetamiprid, tiacloprid, thiamethoxam), nicotinic acetylcholine receptor (nAChR) allosteric modulators, such as spinosyns (e.g., spinose and spinetoram), chloride channel activators, such as avermectins and milbemycins (e.g., abamectin, emamectin benzoate), Nicotinic acetylcholine receptor (nAChR) blockers, such as bensultap and cartap, voltage dependent sodium channel blockers, such as indoxacarb and metaflumizone, ryanodine receptor modulator, such as diamides (e.g. dhlorantraniliprole and flubendiamide). In another embodiment, the insecticides are insecticides that target respiration. Examples include chemicals that uncouple oxidative phosphorylation via disruption of the proton gradient, such as chlorfenapyr, and mitochondrial complex I electron transport inhibitors.
In another embodiment, the insecticides are insecticides that target midgut. Examples include microbial disruptors of insect midgut membranes, such as Bacillus thuringiensis and Bacillus sphaericus.
In another embodiment, the insecticides are insecticides that target growth and development. Examples include juvenile hormone mimics, such as juvenile hormone analogues (e.g. fenoxycarb), inhibitors of chitin biosynthesis, Type 0, such as benzoylureas (e.g., flufenoxuron, lufenuron, and novaluron), and ecdysone receptor agonists, such as diacylhydrazines (e.g., methoxyfenozide and tebufenozide)
Stabilizer
According to another embodiment of the disclosure, the pheromone composition may include one or more additives that enhance the stability of the composition. Examples of additives include, but are not limited to, fatty acids and vegetable oils, such as for example olive oil, soybean oil, corn oil, safflower oil, canola oil, and combinations thereof.
Filler
According to another embodiment of the disclosure, the pheromone composition may include one or more fillers. Examples of fillers include, but are not limited to, one or more mineral clays (e.g., attapulgite). In some embodiments, the attractant-composition may include one or more organic thickeners. Examples of such thickeners include, but are not limited to, methyl cellulose, ethyl cellulose, and any combinations thereof
Solvent
According to another embodiment, the pheromone compositions of the present disclosure can include one or more solvents. Compositions containing solvents are desirable when a user is to employ liquid compositions which may be applied by brushing, dipping, rolling, spraying, or otherwise applying the liquid compositions to substrates on which the user wishes to provide a pheromone coating (e.g., a lure). In some embodiments, the solvent(s) to be used is/are selected so as to solubilize, or substantially solubilize, the one or more ingredients of the pheromone composition. Examples of solvents include, but are not limited to, water, aqueous solvent (e.g., mixture of water and ethanol), ethanol, methanol, chlorinated hydrocarbons, petroleum solvents, turpentine, xylene, and any combinations thereof.
In some embodiments, the pheromone compositions of the present disclosure comprise organic solvents. Organic solvents are used mainly in the formulation of emulsifiable concentrates, ULV formulations, and to a lesser extent granular formulations. Sometimes mixtures of solvents are used. In some embodiments, the present disclosure teaches the use of solvents including aliphatic paraffinic oils such as kerosene or refined paraffins. In other embodiments, the present disclosure teaches the use of aromatic solvents such as xylene and higher molecular weight fractions of C9 and C10 aromatic solvents. In some embodiments, chlorinated hydrocarbons are useful as co-solvents to prevent crystallization when the formulation is emulsified into water. Alcohols are sometimes used as co-solvents to increase solvent power.
Solubilizing Agent
In some embodiments, the pheromone compositions of the present disclosure comprise solubilizing agents. A solubilizing agent is a surfactant, which will form micelles in water at concentrations above the critical micelle concentration. The micelles are then able to dissolve or solubilize water-insoluble materials inside the hydrophobic part of the micelle. The types of surfactants usually used for solubilization are non-ionics: sorbitan monooleates; sorbitan monooleate ethoxylates; and methyl oleate esters.
Binder
According to another embodiment of the disclosure, the pheromone composition may include one or more binders. Binders can be used to promote association of the pheromone composition with the surface of the material on which said composition is coated. In some embodiments, the binder can be used to promote association of another additive (e.g., insecticide, insect growth regulators, and the like) to the pheromone composition and/or the surface of a material. For example, a binder can include a synthetic or natural resin typically used in paints and coatings. These may be modified to cause the coated surface to be friable enough to allow insects to bite off and ingest the components of the composition (e.g., insecticide, insect growth regulators, and the like), while still maintaining the structural integrity of the coating.
Non-limiting examples of binders include polyvinylpyrrolidone, polyvinyl alcohol, partially hydrolyzed polyvinyl acetate, carboxymethylcellulose, starch, vinylpyrrolidone/vinyl acetate copolymers and polyvinyl acetate, or compositions of these; lubricants such as magnesium stearate, sodium stearate, talc or polyethylene glycol, or compositions of these; antifoams such as silicone emulsions, long-chain alcohols, phosphoric esters, acetylene diols, fatty acids or organofluorine compounds, and complexing agents such as: salts of ethylenediaminetetraacetic acid (EDTA), salts of trinitrilotriacetic acid or salts of polyphosphoric acids, or compositions of these.
In some embodiments, the binder also acts a filler and/or a thickener. Examples of such binders include, but are not limited to, one or more of shellac, acrylics, epoxies, alkyds, polyurethanes, linseed oil, tung oil, and any combinations thereof
Surface-Active Agents
In some embodiments, the pheromone compositions comprise surface-active agents. In some embodiments, the surface-active agents are added to liquid agricultural compositions. In other embodiments, the surface-active agents are added to solid formulations, especially those designed to be diluted with a carrier before application. Thus, in some embodiments, the pheromone compositions comprise surfactants. Surfactants are sometimes used, either alone or with other additives, such as mineral or vegetable oils as adjuvants to spray-tank mixes to improve the biological performance of the pheromone on the target. The surface-active agents can be anionic, cationic, or nonionic in character, and can be employed as emulsifying agents, wetting agents, suspending agents, or for other purposes. In some embodiments, the surfactants are non-ionics such as: alky ethoxylates, linear aliphatic alcohol ethoxylates, and aliphatic amine ethoxylates. Surfactants conventionally used in the art of formulation and which may also be used in the present formulations are described, in McCutcheon's Detergents and Emulsifiers Annual, MC Publishing Corp., Ridgewood, N.J., 1998, and in Encyclopedia of Surfactants, Vol. I-III, Chemical Publishing Co., New York, 1980-81. In some embodiments, the present disclosure teaches the use of surfactants including alkali metal, alkaline earth metal or ammonium salts of aromatic sulfonic acids, for example, ligno-, phenol-, naphthalene- and dibutylnaphthalenesulfonic acid, and of fatty acids of arylsulfonates, of alkyl ethers, of lauryl ethers, of fatty alcohol sulfates and of fatty alcohol glycol ether sulfates, condensates of sulfonated naphthalene and its derivatives with formaldehyde, condensates of naphthalene or of the naphthalenesulfonic acids with phenol and formaldehyde, condensates of phenol or phenolsulfonic acid with formaldehyde, condensates of phenol with formaldehyde and sodium sulfite, polyoxyethylene octylphenyl ether, ethoxylated isooctyl-, octyl- or nonylphenol, tributylphenyl polyglycol ether, alkylaryl polyether alcohols, isotridecyl alcohol, ethoxylated castor oil, ethoxylated triarylphenols, salts of phosphated triarylphenolethoxylates, lauryl alcohol polyglycol ether acetate, sorbitol esters, lignin-sulfite waste liquors or methylcellulose, or compositions of these.
In some embodiments, the present disclosure teaches other suitable surface-active agents, including salts of alkyl sulfates, such as diethanolammonium lauryl sulfate; alkylarylsulfonate salts, such as calcium dodecylbenzenesulfonate; alkylphenol-alkylene oxide addition products, such as nonylphenol-C18 ethoxylate; alcohol-alkylene oxide addition products, such as tridecyl alcohol-C16 ethoxylate; soaps, such as sodium stearate; alkylnaphthalene-sulfonate salts, such as sodium dibutyl-naphthalenesulfonate; dialkyl esters of sulfosuccinate salts, such as sodium di(2-ethylhexyl)sulfosuccinate; sorbitol esters, such as sorbitol oleate; quaternary amines, such as lauryl trimethylammonium chloride; polyethylene glycol esters of fatty acids, such as polyethylene glycol stearate; block copolymers of ethylene oxide and propylene oxide; salts of mono and dialkyl phosphate esters; vegetable oils such as soybean oil, rapeseed/canola oil, olive oil, castor oil, sunflower seed oil, coconut oil, corn oil, cottonseed oil, linseed oil, palm oil, peanut oil, safflower oil, sesame oil, tung oil and the like; and esters of the above vegetable oils, particularly methyl esters.
Wetting Agents
In some embodiments, the pheromone compositions comprise wetting agents. A wetting agent is a substance that when added to a liquid increases the spreading or penetration power of the liquid by reducing the interfacial tension between the liquid and the surface on which it is spreading. Wetting agents are used for two main functions in agrochemical formulations: during processing and manufacture to increase the rate of wetting of powders in water to make concentrates for soluble liquids or suspension concentrates; and during mixing of a product with water in a spray tank or other vessel to reduce the wetting time of wettable powders and to improve the penetration of water into water-dispersible granules. In some embodiments, examples of wetting agents used in the pheromone compositions of the present disclosure, including wettable powders, suspension concentrates, and water-dispersible granule formulations are: sodium lauryl sulphate; sodium dioctyl sulphosuccinate; alkyl phenol ethoxylates; and aliphatic alcohol ethoxylates.
Dispersing Agent
In some embodiments, the pheromone compositions of the present disclosure comprise dispersing agents. A dispersing agent is a substance which adsorbs onto the surface of particles and helps to preserve the state of dispersion of the particles and prevents them from reaggregating. In some embodiments, dispersing agents are added to pheromone compositions of the present disclosure to facilitate dispersion and suspension during manufacture, and to ensure the particles redisperse into water in a spray tank. In some embodiments, dispersing agents are used in wettable powders, suspension concentrates, and water-dispersible granules. Surfactants that are used as dispersing agents have the ability to adsorb strongly onto a particle surface and provide a charged or steric barrier to re-aggregation of particles. In some embodiments, the most commonly used surfactants are anionic, non-ionic, or mixtures of the two types.
In some embodiments, for wettable powder formulations, the most common dispersing agents are sodium lignosulphonates. In some embodiments, suspension concentrates provide very good adsorption and stabilization using polyelectrolytes, such as sodium naphthalene sulphonate formaldehyde condensates. In some embodiments, tristyrylphenol ethoxylated phosphate esters are also used. In some embodiments, such as alkylarylethylene oxide condensates and EO-PO block copolymers are sometimes combined with anionics as dispersing agents for suspension concentrates.
Polymeric Surfactant
In some embodiments, the pheromone compositions of the present disclosure comprise polymeric surfactants. In some embodiments, the polymeric surfactants have very long hydrophobic ‘backbones’ and a large number of ethylene oxide chains forming the ‘teeth’ of a ‘comb’ surfactant. In some embodiments, these high molecular weight polymers can give very good long-term stability to suspension concentrates, because the hydrophobic backbones have many anchoring points onto the particle surfaces. In some embodiments, examples of dispersing agents used in pheromone compositions of the present disclosure are: sodium lignosulphonates; sodium naphthalene sulphonate formaldehyde condensates; tristyrylphenol ethoxylate phosphate esters; aliphatic alcohol ethoxylates; alky ethoxylates; EO-PO block copolymers; and graft copolymers.
Emulsifying Agent
In some embodiments, the pheromone compositions of the present disclosure comprise emulsifying agents. An emulsifying agent is a substance, which stabilizes a suspension of droplets of one liquid phase in another liquid phase. Without the emulsifying agent the two liquids would separate into two immiscible liquid phases. In some embodiments, the most commonly used emulsifier blends include alkylphenol or aliphatic alcohol with 12 or more ethylene oxide units and the oil-soluble calcium salt of dodecylbenzene sulphonic acid. A range of hydrophile-lipophile balance (“HLB”) values from 8 to 18 will normally provide good stable emulsions. In some embodiments, emulsion stability can sometimes be improved by the addition of a small amount of an EO-PO block copolymer surfactant.
Gelling Agent
In some embodiments, the pheromone compositions comprise gelling agents. Thickeners or gelling agents are used mainly in the formulation of suspension concentrates, emulsions, and suspoemulsions to modify the rheology or flow properties of the liquid and to prevent separation and settling of the dispersed particles or droplets. Thickening, gelling, and anti-settling agents generally fall into two categories, namely water-insoluble particulates and water-soluble polymers. It is possible to produce suspension concentrate formulations using clays and silicas. In some embodiments, the pheromone compositions comprise one or more thickeners including, but not limited to: montmorillonite, e.g. bentonite; magnesium aluminum silicate; and attapulgite. In some embodiments, the present disclosure teaches the use of polysaccharides as thickening agents. The types of polysaccharides most commonly used are natural extracts of seeds and seaweeds or synthetic derivatives of cellulose. Some embodiments utilize xanthan and some embodiments utilize cellulose. In some embodiments, the present disclosure teaches the use of thickening agents including, but are not limited to: guar gum; locust bean gum; carrageenam; alginates; methyl cellulose; sodium carboxymethyl cellulose (SCMC); hydroxyethyl cellulose (HEC). In some embodiments, the present disclosure teaches the use of other types of anti-settling agents such as modified starches, polyacrylates, polyvinyl alcohol, and polyethylene oxide. Another good anti-settling agent is xanthan gum.
Anti-Foam Agent
In some embodiments, the presence of surfactants, which lower interfacial tension, can cause water-based formulations to foam during mixing operations in production and in application through a spray tank. Thus, in some embodiments, in order to reduce the tendency to foam, anti-foam agents are often added either during the production stage or before filling into bottles/spray tanks. Generally, there are two types of anti-foam agents, namely silicones and nonsilicones. Silicones are usually aqueous emulsions of dimethyl polysiloxane, while the nonsilicone anti-foam agents are water-insoluble oils, such as octanol and nonanol, or silica. In both cases, the function of the anti-foam agent is to displace the surfactant from the air-water interface.
Preservative
In some embodiments, the pheromone compositions comprise a preservative.
Additional Active Agent
According to another embodiment of the disclosure, the pheromone composition may include one or more insect feeding stimulants. Examples of insect feeding stimulants include, but are not limited to, crude cottonseed oil, fatty acid esters of phytol, fatty acid esters of geranyl geraniol, fatty acid esters of other plant alcohols, plant extracts, and combinations thereof.
According to another embodiment of the disclosure, the pheromone composition may include one or more insect growth regulators (“IGRs”). IGRs may be used to alter the growth of the insect and produce deformed insects. Examples of insect growth regulators include, for example, dimilin.
According to another embodiment of the disclosure, the attractant-composition may include one or more insect sterilants that sterilize the trapped insects or otherwise block their reproductive capacity, thereby reducing the population in the following generation. In some situations allowing the sterilized insects to survive and compete with non-trapped insects for mates is more effective than killing them outright.
Sprayable Compositions
In some embodiments, the pheromone compositions disclosed herein can be formulated as a sprayable composition (i.e., a sprayable pheromone composition). An aqueous solvent can be used in the sprayable composition, e.g., water or a mixture of water and an alcohol, glycol, ketone, or other water-miscible solvent. In some embodiments, the water content of such mixture is at least about 10%, at least about 20%, at least about 30%, at least about 40%, 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%. In some embodiments, the sprayable composition is concentrate, i.e. a concentrated suspension of the pheromone, and other additives (e.g., a waxy substance, a stabilizer, and the like) in the aqueous solvent, and can be diluted to the final use concentration by addition of solvent (e.g., water).
In some embodiments, a waxy substance can be used as a carrier for the pheromone and its positional isomer in the sprayable composition. The waxy substance can be, e.g., a biodegradable wax, such as bees wax, carnauba wax and the like, candelilla wax (hydrocarbon wax), montan wax, shellac and similar waxes, saturated or unsaturated fatty acids, such as lauric, palmitic, oleic or stearic acid, fatty acid amides and esters, hydroxylic fatty acid esters, such as hydroxyethyl or hydroxypropyl fatty acid esters, fatty alcohols, and low molecular weight polyesters such as polyalkylene succinates.
In some embodiments, a stabilizer can be used with the sprayable pheromone compositions. The stabilizer can be used to regulate the particle size of concentrate and/or to allow the preparation of a stable suspension of the pheromone composition. In some embodiments, the stabilizer is selected from hydroxylic and/or ethoxylated polymers. Examples include ethylene oxide and propylene oxide copolymer, polyalcohols, including starch, maltodextrin and other soluble carbohydrates or their ethers or esters, cellulose ethers, gelatin, polyacrylic acid and salts and partial esters thereof and the like. In other embodiments, the stabilizer can include polyvinyl alcohols and copolymers thereof, such as partly hydrolyzed polyvinyl acetate. The stabilizer may be used at a level sufficient to regulate particle size and/or to prepare a stable suspension, e.g., between 0.1% and 15% of the aqueous solution.
In some embodiments, a binder can be used with the sprayable pheromone compositions. In some embodiments, the binder can act to further stabilize the dispersion and/or improve the adhesion of the sprayed dispersion to the target locus (e.g., trap, lure, plant, and the like). The binder can be polysaccharide, such as an alginate, cellulose derivative (acetate, alkyl, carboxymethyl, hydroxyalkyl), starch or starch derivative, dextrin, gum (arabic, guar, locust bean, tragacanth, carrageenan, and the like), sucrose, and the like. The binder can also be a non-carbohydrate, water-soluble polymer such as polyvinyl pyrrolidone, or an acidic polymer such as polyacrylic acid or polymethacrylic acid, in acid and/or salt form, or mixtures of such polymers.
Microencapsulated Pheromones
In some embodiments, the pheromone compositions disclosed herein can be formulated as a microencapsulated pheromone, such as disclosed in Ill'lchev, A L et al., J. Econ. Entomol. 2006; 99(6):2048-54; and Stelinki, L L et al., J. Econ. Entomol. 2007; 100(4):1360-9. Microencapsulated pheromones (MECs) are small droplets of pheromone enclosed within polymer capsules. The capsules control the release rate of the pheromone into the surrounding environment, and are small enough to be applied in the same method as used to spray insecticides. The effective field longevity of the microencapsulated pheromone formulations can range from a few days to slightly more than a week, depending on inter alia climatic conditions, capsule size and chemical properties.
Slow-Release Formulation
Pheromone compositions can be formulated so as to provide slow release into the atmosphere, and/or so as to be protected from degradation following release. For example, the pheromone compositions can be included in carriers such as microcapsules, biodegradable flakes and paraffin wax-based matrices. Alternatively, the pheromone composition can be formulated as a slow release sprayable.
In certain embodiments, the pheromone composition may include one or more polymeric agents known to one skilled in the art. The polymeric agents may control the rate of release of the composition to the environment. In some embodiments, the polymeric attractant-composition is impervious to environmental conditions. The polymeric agent may also be a sustained-release agent that enables the composition to be released to the environment in a sustained manner.
Examples of polymeric agents include, but are not limited to, celluloses, proteins such as casein, fluorocarbon-based polymers, hydrogenated rosins, lignins, melamine, polyurethanes, vinyl polymers such as polyvinyl acetate (PVAC), polycarbonates, polyvinylidene dinitrile, polyamides, polyvinyl alcohol (PVA), polyamide-aldehyde, polyvinyl aldehyde, polyesters, polyvinyl chloride (PVC), polyethylenes, polystyrenes, polyvinylidene, silicones, and combinations thereof. Examples of celluloses include, but are not limited to, methylcellulose, ethyl cellulose, cellulose acetate, cellulose acetate-butyrate, cellulose acetate-propionate, cellulose propionate, and combinations thereof.
Other agents which can be used in slow-release or sustained-release formulations include fatty acid esters (such as a sebacate, laurate, palmitate, stearate or arachidate ester) or a fatty alcohols (such as undecanol, dodecanol, tridecanol, tridecenol, tetradecanol, tetradecenol, tetradecadienol, pentadecanol, pentadecenol, hexadecanol, hexadecenol, hexadecadienol, octadecenol and octadecadienol).
Pheromones prepared according to the methods of the invention, as well as compositions containing the pheromones, can be used to control the behavior and/or growth of insects in various environments. The pheromones can be used, for example, to attract or repel male or female insects to or from a particular target area. The pheromones can be used to attract insects away from vulnerable crop areas. The pheromones can also be used example to attract insects as part of a strategy for insect monitoring, mass trapping, lure/attract-and-kill or mating disruption.
Lures
The pheromone compositions of the present disclosure may be coated on or sprayed on a lure, or the lure may be otherwise impregnated with a pheromone composition.
Traps
The pheromone compositions of the disclosure may be used in traps, such as those commonly used to attract any insect species, e.g., insects of the order Lepidoptera. Such traps are well known to one skilled in the art, and are commonly used in many states and countries in insect eradication programs. In one embodiment, the trap includes one or more septa, containers, or storage receptacles for holding the pheromone composition. Thus, in some embodiments, the present disclosure provides a trap loaded with at least one pheromone composition. Thus, the pheromone compositions of the present disclosure can be used in traps for example to attract insects as part of a strategy for insect monitoring, mass trapping, mating disruption, or lure/attract and kill for example by incorporating a toxic substance into the trap to kill insects caught.
Mass trapping involves placing a high density of traps in a crop to be protected so that a high proportion of the insects are removed before the crop is damaged. Lure/attract-and-kill techniques are similar except once the insect is attracted to a lure, it is subjected to a killing agent. Where the killing agent is an insecticide, a dispenser can also contain a bait or feeding stimulant that will entice the insects to ingest an effective amount of an insecticide. The insecticide may be an insecticide known to one skilled in the art. The insecticide may be mixed with the attractant-composition or may be separately present in a trap. Mixtures may perform the dual function of attracting and killing the insect.
Such traps may take any suitable form, and killing traps need not necessarily incorporate toxic substances, the insects being optionally killed by other means, such as drowning or electrocution. Alternatively, the traps can contaminate the insect with a fungus or virus that kills the insect later. Even where the insects are not killed, the trap can serve to remove the male insects from the locale of the female insects, to prevent breeding.
It will be appreciated by a person skilled in the art that a variety of different traps are possible. Suitable examples of such traps include water traps, sticky traps, and one-way traps. Sticky traps come in many varieties. One example of a sticky trap is of cardboard construction, triangular or wedge-shaped in cross-section, where the interior surfaces are coated with a non-drying sticky substance. The insects contact the sticky surface and are caught. Water traps include pans of water and detergent that are used to trap insects. The detergent destroys the surface tension of the water, causing insects that are attracted to the pan, to drown in the water. One-way traps allow an insect to enter the trap but prevent it from exiting. The traps of the disclosure can be colored brightly, to provide additional attraction for the insects.
In some embodiments, the pheromone traps containing the composition may be combined with other kinds of trapping mechanisms. For example, in addition to the pheromone composition, the trap may include one or more florescent lights, one or more sticky substrates and/or one or more colored surfaces for attracting moths. In other embodiments, the pheromone trap containing the composition may not have other kinds of trapping mechanisms.
The trap may be set at any time of the year in a field. Those of skill in the art can readily determine an appropriate amount of the compositions to use in a particular trap, and can also determine an appropriate density of traps/acre of crop field to be protected.
The trap can be positioned in an area infested (or potentially infested) with insects. Generally, the trap is placed on or close to a tree or plant. The aroma of the pheromone attracts the insects to the trap. The insects can then be caught, immobilized and/or killed within the trap, for example, by the killing agent present in the trap.
Traps may also be placed within an orchard to overwhelm the pheromones emitted by the females, so that the males simply cannot locate the females. In this respect, a trap need be nothing more than a simple apparatus, for example, a protected wickable to dispense pheromone.
The traps of the present disclosure may be provided in made-up form, where the compound of the disclosure has already been applied. In such an instance, depending on the half-life of the compound, the compound may be exposed, or may be sealed in conventional manner, such as is standard with other aromatic dispensers, the seal only being removed once the trap is in place.
Alternatively, the traps may be sold separately, and the compound of the disclosure provided in dispensable format so that an amount may be applied to trap, once the trap is in place. Thus, the present disclosure may provide the compound in a sachet or other dispenser.
Dispenser
Pheromone compositions can be used in conjunction with a dispenser for release of the composition in a particular environment. Any suitable dispenser known in the art can be used. Examples of such dispensers include but are not limited to, aerosol emitters, hand-applied dispensers, bubble caps comprising a reservoir with a permeable barrier through which pheromones are slowly released, pads, beads, tubes rods, spirals or balls composed of rubber, plastic, leather, cotton, cotton wool, wood or wood products that are impregnated with the pheromone composition. For example, polyvinyl chloride laminates, pellets, granules, ropes or spirals from which the pheromone composition evaporates, or rubber septa. One of skill in the art will be able to select suitable carriers and/or dispensers for the desired mode of application, storage, transport or handling.
In another embodiment, a device may be used that contaminates the male insects with a powder containing the pheromone substance itself. The contaminated males then fly off and provide a source of mating disruption by permeating the atmosphere with the pheromone substance, or by attracting other males to the contaminated males, rather than to real females.
Behavior Modification
Pheromone compositions prepared according to the methods disclosed herein can be used to control or modulate the behavior of insects. In some embodiments, the behavior of the target insect can be modulated in a tunable manner inter alia by varying the ratio of the pheromone to the positional isomer in the composition such that the insect is attracted to a particular locus but does not contact said locus or such the insect in fact contacts said locus. Thus, in some embodiments, the pheromones can be used to attract insects away from vulnerable crop areas. Accordingly, the disclosure also provides a method for attracting insects to a locus. The method includes administering to a locus an effective amount of the pheromone composition.
The method of mating disruption may include periodically monitoring the total number or quantity of the trapped insects. The monitoring may be performed by counting the number of insects trapped for a predetermined period of time such as, for example, daily, Weekly, bi-Weekly, monthly, once-in-three months, or any other time periods selected by the monitor. Such monitoring of the trapped insects may help estimate the population of insects for that particular period, and thereby help determine a particular type and/or dosage of pest control in an integrated pest management system. For example, a discovery of a high insect population can necessitate the use of methods for removal of the insect. Early warning of an infestation in a new habitat can allow action to be taken before the population becomes unmanageable. Conversely, a discovery of a low insect population can lead to a decision that it is sufficient to continue monitoring the population. Insect populations can be monitored regularly so that the insects are only controlled when they reach a certain threshold. This provides cost-effective control of the insects and reduces the environmental impact of the use of insecticides.
Mating Disruption
Pheromones prepared according to the methods of the disclosure can also be used to disrupt mating. Mating disruption is a pest management technique designed to control insect pests by introducing artificial stimuli (e.g., a pheromone composition as disclosed herein) that confuses the insects and disrupts mating localization and/or courtship, thereby preventing mating and blocking the reproductive cycle.
In many insect species of interest to agriculture, such as those in the order Lepidoptera, females emit an airborne trail of a specific chemical blend constituting that species' sex pheromone. This aerial trail is referred to as a pheromone plume. Males of that species use the information contained in the pheromone plume to locate the emitting female (known as a “calling” female). Mating disruption exploits the male insects' natural response to follow the plume by introducing a synthetic pheromone into the insects' habitat, which is designed to mimic the sex pheromone produced by the female insect. Thus, in some embodiments, the synthetic pheromone utilized in mating disruption is a synthetically derived pheromone composition comprising a pheromone having a chemical structure of a sex pheromone and a positional isomer thereof which is not produced by the target insect.
The general effect of mating disruption is to confuse the male insects by masking the natural pheromone plumes, causing the males to follow “false pheromone trails” at the expense of finding mates, and affecting the males' ability to respond to “calling” females. Consequently, the male population experiences a reduced probability of successfully locating and mating with females, which leads to the eventual cessation of breeding and collapse of the insect infestation
Strategies of mating disruption include confusion, trail-masking and false-trail following. Constant exposure of insects to a high concentration of a pheromone can prevent male insects from responding to normal levels of the pheromone released by female insects. Trail-masking uses a pheromone to destroy the trail of pheromones released by females. False-trail following is carried out by laying numerous spots of a pheromone in high concentration to present the male with many false trails to follow. When released in sufficiently high quantities, the male insects are unable to find the natural source of the sex pheromones (the female insects) so that mating cannot occur.
In some embodiments, a wick or trap may be adapted to emit a pheromone for a period at least equivalent to the breeding season(s) of the midge, thus causing mating disruption. If the midge has an extended breeding season, or repeated breeding season, the present disclosure provides a wick or trap capable of emitting pheromone for a period of time, especially about two weeks, and generally between about 1 and 4 weeks and up to 6 weeks, which may be rotated or replaced by subsequent similar traps. A plurality of traps containing the pheromone composition may be placed in a locus, e.g., adjacent to a crop field. The locations of the traps, and the height of the traps from ground may be selected in accordance with methods known to one skilled in the art.
Alternatively, the pheromone composition may be dispensed from formulations such as microcapsules or twist-ties, such as are commonly used for disruption of the mating of insect pests.
Attract and Kill
The attract and kill method utilizes an attractant, such as a sex pheromone, to lure insects of the target species to an insecticidal chemical, surface, device, etc., for mass-killing and ultimate population suppression, and can have the same effect as mass-trapping. For instance, when a synthetic female sex pheromone is used to lure male pests, e.g., moths, in an attract-and-kill strategy, a large number of male moths must be killed over extended periods of time to reduce matings and reproduction, and ultimately suppress the pest population. The attract-and-kill approach may be a favorable alternative to mass-trapping because no trap-servicing or other frequent maintenance is required. In various embodiments described herein, a recombinant microorganism can co-express (i) a pathway for production of an insect pheromone and (ii) a protein, peptide, oligonucleotide, or small molecule which is toxic to the insect. In this way, the recombinant microorganism can co-produce substances suitable for use in an attract-and-kill approach.
As will be apparent to one of skill in the art, the amount of a pheromone or pheromone composition used for a particular application can vary depending on several factors such as the type and level of infestation; the type of composition used; the concentration of the active components; how the composition is provided, for example, the type of dispenser used; the type of location to be treated; the length of time the method is to be used for; and environmental factors such as temperature, wind speed and direction, rainfall and humidity. Those of skill in the art will be able to determine an effective amount of a pheromone or pheromone composition for use in a given application.
As used herein, an “effective amount” means that amount of the disclosed pheromone composition that is sufficient to affect desired results. An effective amount can be administered in one or more administrations. For example, an effective amount of the composition may refer to an amount of the pheromone composition that is sufficient to attract a given insect to a given locus. Further, an effective amount of the composition may refer to an amount of the pheromone composition that is sufficient to disrupt mating of a particular insect population of interest in a given locality.
EXAMPLES Example 1 Production of Pheromones Products from Enzymatically-Derived Gondoic Acid through Metathesis and Chemical ConversionThis example illustrates that different fatty acids can be used as a starting material for the biosynthetic production of a pheromone or pheromone precursor. The product obtained from the biosynthetic process disclosed herein can be subject to further chemical conversions to generate different products.
Enzymatic two carbon elongation of oleic acid yields gondoic acid. After esterification, gondoic fatty acid methyl ester (FAME) can then converted via Z-selective olefin metathesis into C16 and C18 FAME products containing a C11 unsaturation. Upon reduction of the ester, aldehyde and fatty alcohol pheromone materials can be produced. Acetylation of the fatty alcohol product can generate the corresponding fatty acetate pheromones. Additionally, gondoic acid can be directly converted into C20 fatty aldehyde, alcohol and acetate pheromones through application of the same chemical transformation of enzymatically modified oleic acid.
Prophetic Example 2 Tailored Synthetic BlendsThis prophetic example illustrates that the recombinant microorganisms disclosed herein can be used to create synthetic blends of insect pheromones.
As shown in the scheme depicted in
Similarly, using hexadecyl-ACP (16:ACP), a blend of Z- and E hexadecenyl acetate pheromones (E11-16:OAc and Z11-16:OAc) can be produced with the recombinant microorganism.
The microorganism can be engineered with different desaturases, or other enzymes such as reductases, etc. to produce the desired blend of pheromones. One blend of particular relevance capable of being produced using the recombinant microorganisms and methods of the instant invention is a 97:3 ratio of (Z)-11-hexadecenal (Z11-16:Ald) and (Z)-9-hexadecenal (Z9-16:Ald).
Example 3 Expression of Transmembrane Alcohol-Forming Reductases in S. cerevisiae Background and RationaleEngineering microbial production of insect fatty alcohols from fatty acids entails the functional expression of a synthetic pathway. One such pathway comprises a transmembrane desaturase, and an alcohol-forming reductase to mediate the conversion of fatty acyl-CoA into regio- and stereospecific unsaturated fatty acyl-CoA, and subsequently into fatty alcohols. A number of genes encoding these enzymes are found in some insects (as well as some microalgae in the case of fatty alcohol reductase) and can be used to construct the synthetic pathway in yeasts, which are preferred production hosts. A number of transmembrane desaturases and alcohol-forming reductase variants will be screened to identify ensembles which allow high level synthesis of a single insect fatty alcohol or a blend of fatty alcohols. Additionally, these enzymes will be screened across multiple hosts (Saccharomyces cerevisiae, Candida tropicalis, and Yarrowia lipolytica) to optimize the search toward finding a suitable host for optimum expression of these transmembrane proteins.
Summary of Approach
Three alcohol-forming reductases of insect origin were selected.
Nucleic acids encoding the reductases were synthesized (synthons) with codon optimization for expression in S. cerevisiae.
Each nucleic acid encoding a given reductase was subcloned into an episomal expression cassette under the Gal1 promoter.
S. cerevisiae wild-type and beta-oxidation deletion mutant were transformed with expression constructs.
Heterologous protein was induced by galactose, and functional expression of the reductases was assessed in vivo via bioconversion of Z11-hexadecenoic acid into Z11-hexedecenol.
GC-MS analysis was used to identify and quantify metabolites.
Results
Alcohol-forming reductase variants were screened for activity in S. cerevisiae W303 (wild type) and BY4742 ΔPOX1 (beta-oxidation deletion mutant). Z11-hexadecenoic acid was chosen as a substrate in assessing enzyme activity. The in vivo bioconversion assay showed that the expression of enzyme variants derived from Spodoptera littoralis, Helicoverpa armigera, and Agrotis segetum (Ding, B-J., Löfstedt, C. Analysis of the Agrotis segetum pheromone gland transcriptome in the light of sex pheromone biosynthesis. BMC Genomics 16:711 (2015)) in W303A conferred Z11-hexadecenol production, and reached up-to ˜37 μM (8 mg/L), ˜70 μM (˜16 mg/L), and 11 μM (˜3 mg/L), respectively, within 48 h of protein induction (
Therefore, functional expression of at least two alcohol-forming reductases in S. cerevisiae conferred bioconversion of Z11-hexadecenoic acid into Z11-hexedecenol.
Conclusions
Functional expression of insect transmembrane alcohol-forming reductase in S. cerevisiae was demonstrated. Among the reductases tested, the variant derived from Helicoverpa armigera is most active toward Z11-hexadecenoic acid.
The bioconversion of other fatty acid substrates can be explored to assess enzyme plasticity.
Materials & Methods
Strain Construction and Functional Expression Assay
S. cerevisiae W303 (MATA ura3-1 trp1-1 leu2-3_112 his3-11_15 ade2-1 can1-100) and BY4742 (MATa POX1::kanMX his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0) were used as expression hosts. DNA sequences which encode fatty alcohol reductase variants were redesigned to optimize expression in S. cerevisiae (SEQ ID NOs: 1-3). Generated synthons (Genscript) were cloned into pESC-URA vector using BamHI-XhoI sites to facilitate protein expression utilizing the Gal1 promoter. The resulting plasmid constructs were used to transform W303, and positive transformants were selected on CM agar medium (with 2% glucose, and lacking uracil) (Teknova). To assess functional expression, two positive transformation clones that have been patched on CM agar medium (with 2% glucose, and lacking uracil) were used to seed CM liquid medium using a 24 deep-well plate format. To induce protein expression, the overnight cultures that had been grown at 28° C. were then supplemented with galactose, raffinose, and YNB to a final concentration of 2%, 1%, and 6.7 g/L, respectively. Post 24 h of protein induction, the bioconversion substrate Z11-hexadecenoic acid (in ethanol) or heptadecanoic acid (in ethanol) was added to a final concentration of 300 mg/L. Bioconversion assay proceeded for 48 h at 28° C. prior to GC-MS analysis.
Metabolite Extraction and GC-MS Detection
The lipids were extracted according to a modified procedure of Hagström et al. (2012) (Hagström, A. K., Liénard, M. A., Groot, A. T., Hedenström, E. & Löfstedt, C. Semi-Selective Fatty Acyl Reductases from Four Heliothine Moths Influence the Specific Pheromone Composition. PLoS One 7: e37230 (2012)). 1.5 mL-cell culture was transferred to a 15 mL falcon tube. The cell suspension was acidified with 1 mL 5 N HCl. 5 μL tetradecanedioic acid (10 mM in ethanol) was added as internal standard. The mixture was extracted by adding 1.5 mL hexane, then shaken for 1 h at 37° C., 250 rpm. To facilitate phase separation, the sample was centrifuged for 10 min at 2000 g. 1 mL of the organic hexane phase was then transferred to a 1.5 mL plastic tube. The solvent was removed by heating the sample 30 min at 90° C. After the sample was evaporated to dryness, 50 μL of BSTFA (N,O-bis(trimethylsilyl) trifluoroacetamide containing 1% of trimethylchlorosilane) was added. The 1.5 mL plastic tubes were shaken vigorously two times for 10 s. Prior to the transfer into a screw cap GC glass vial containing a glass insert, the sample was centrifuged for 1 min (13000 rpm). The vials were capped and heated for 30 min at 90° C. The trimethylsilyl-esters, which were generated by this method were subsequently analyzed by GC-MS analysis. GC-MS parameters are specified in Table 6. The use of SIM mode (characteristic product and IS ions) increases detection sensitivity by reducing background noise, allowing detection of the product as low as 2.4 μM (0.6 mg/L). A further reduction in the split ratio offers the possibility to further increase the sensitivity for future applications. A Z11-hexadecenol calibration curve shown in
Background and Rationale
Engineering microbial production of insect fatty alcohols from fatty acids requires the functional expression of a synthetic pathway. One such pathway comprises a transmembrane desaturase, and an alcohol-forming reductase to mediate the conversion of fatty acyl-CoA into regio- and stereospecific unsaturated fatty acyl-CoA, and subsequently into fatty alcohols. A number of genes encoding these enzymes are found in some insects as well as some microalgae. A number of transmembrane desaturases and alcohol-forming reductase variants will be screened to identify ensembles which allow high level synthesis of a single insect fatty alcohol or a blend of fatty alcohols. Additionally, these enzymes will be screened across multiple hosts (Saccharomyces cerevisiae, Candida tropicalis, and Yarrowia lipolytica) to optimize the search toward finding a suitable host for optimum expression of these transmembrane proteins.
Summary of Approach
A small set of desaturases (insect origin: Agrotis segetum, Trichoplusia ni, Amyelois transitella, Helicoverpa zea, and marine diatom: Thalassiosira pseudonana) were selected as a test case to explore and establish functional expression assays, metabolite extraction methods, and analytical chemistry.
A synthetic cassette for expression of the desaturases in S. cerevisiae was constructed. The cassette consists of the OLE1 promoter region, OLE1 N-terminal leader sequence, and VSP13 terminator.
The expression cassette was tested for functionality via expression of a GFP variant. Validation of the cassette allowed its utilization for exploring expression of insect desaturase.
S. cerevisiae ΔOLE1 was transformed with expression constructs containing heterologous desaturases. Functionality of the desaturases was assessed via the ability to rescue growth of ΔOLE1 without exogenous supplementation of unsaturated fatty acid (UFA). S. cerevisiae desaturase (OLE1) was used as a positive control of successful complementation.
Functionality of the desaturase was validated via an in vivo bioconversion of hexadecanoic acid (palmitic acid) into (Z)-11-hexadecenoic acid (palmitvaccenic acid).
GC-MS analysis was used to identify and quantify metabolites.
Results
Transmembrane desaturase variants were screened in S. cerevisiae. Three variants were initially tested to explore and establish functional expression assays, metabolite extraction methods, and analytical chemistry. To allow functional expression of these desaturases in S. cerevisiae, an episomal synthetic expression cassette termed pOLE1 cassette (
Functional expression of the heterologous desaturases was further characterized via in vivo bioconversion of palmitic acid into insect-specific UFA. Post ˜96 h-cultivation in minimal medium containing palmitic acid, total fatty acid analysis of S. cerevisiae ΔOLE1 expressing T. ni desaturase revealed production of a new fatty acid species (Z)-11-hexadecenoic acid that is not present in the control strain which expresses native yeast OLE1 desaturase (
In summary, at least three insect desaturases capable of rescuing growth of S. cerevisiae ΔOLE1 without exogenous supplementation of UFA, i.e. (Z)-9-hexadecenoic acid (palmitoleic acid), were identified.
The extent of growth on rich medium (YPD) of S. cerevisiae ΔOLE1 bearing the expression construct was in the following order of desaturase content: OLE1, T. ni, T. pseudonana, and A. segetum.
The extent of growth on minimal medium (CM Glucose w/out uracil) of S. cerevisiae ΔOLE1 bearing the expression construct was in the following order of desaturase content: OLE1, T. ni.
Complementation assays using A. transitella and H. zea desaturases were also done, demonstrating functional expression in Candida tropicalis shown via in vivo bioconversion assay. These desaturases also complemented S. cerevisiae ΔOLE1 growth on rich and minimal media at least as well as T. ni desaturase.
Expression of T. pseudonana and A. segetum desaturases did not confer growth of S. cerevisiae ΔOLE1 on minimal medium without UFAs even after an extended incubation period up to 14 days. No (Z)-11-hexadecenoic acid was observed in strains harboring T. pseudonana or A. segetum desaturase.
Conclusions
Functional expression of transmembrane desaturases of insect origin in S. cerevisiae has been achieved.
The activity of a given heterologous desaturase can be assessed from its ability to complement growth of S. cerevisiae ΔOLE1 without exogenous palmitoleic supplementation, and its ability to convert palmitic acid into insect pheromone precursors (Z)-11-hexadecenoic acid.
Functional expression and/or activity of insect desaturase in S. cerevisiae varies widely depending on sequence origin. Variants derived from T. ni exhibited the best activity compared to A. segetum and T. pseudonana, as measured by the above criteria.
Desaturases derived from A. transitella and H. zea complemented ΔOLE1 as well as T. ni desaturase. Bioconversion assays using these desaturases can be done.
The bioconversion of other fatty acid substrates can be explored to assess enzyme plasticity.
Materials & Methods
Strain Construction and Functional Expression Assay
S. cerevisiae ΔOLE1 (MATA OLE1::LEU2 ura3-52 his4) was used as an expression host. A synthetic expression cassette termed pOLE1 (
To assess functional expression, two positive transformation clones that had been patched on CM-Ura glucose agar medium containing UFA were inoculated in 1.5 mL CM-Ura glucose liquid medium containing palmitic acid (in ethanol) at a final concentration of 300 mg/L, and with 6.7 g/L of YNB. For (z)-11-hexadecenoic isomer confirmation, a 20 mL culture was generated. Bioconversion assay proceeded for 96 h at 28° C. prior to GC-MS analysis.
Metabolite Extraction and GC-MS Detection
Total lipid composition as well as the (Z)-11-hexadecenoic acid quantification was based on modified procedures by Moss et al. (1982) (Moss, C. W., Shinoda, T. & Samuels, J. W. Determination of cellular fatty acid compositions of various yeasts by gas-liquid chromatography. J. Clin. Microbiol. 16: 1073-1079 (1982)) and Yousuf et al (2010) (Yousuf, A., Sannino, F., Addorisio, V. & Pirozzi, D. Microbial Conversion of Olive Oil Mill Wastewaters into Lipids Suitable for Biodiesel Production. J. Agric. Food Chem. 58: 8630-8635 (2010)). The pelleted cells (in 1.5 mL plastic tubes), usually about 10 mg to 80 mg, were resuspended in methanol containing 5% (w/w) of sodium hydroxide. The alkaline cell suspension was transferred into a 1.8 mL screw-cap GC-vial. The mixture was heated for 1 h in the heat block at 90° C. Prior to acidification with 400 2.5 N HCl the vial was allowed to cool to room temperature. 500 μL chloroform containing 1 mM heptadecanoic were added and the mixture was shaken vigorously, then both aqueous and organic phase were transferred into a 1.5 mL plastic tube. The mixture was centrifuged at 13,000 rpm, afterwards 450 μL of the organic phase were transferred into a new 1.5 mL plastic tube. The aqueous phase was extracted a second time with 500 μL chloroform, this time without heptadecanoic acid. The combined organic phases were evaporated at 90° C. After cooling to room temperature, residual fatty acid methyl esters and free fatty acids were dissolved and derivatized in methanol containing 0.2 M TMSH (trimethylsulfonium hydroxide).
The regioselectivity of biologically produced (Z)-11-hexadecenoic acid was determined by comparing the fragmentation patterns of the dimethyl disulfide (DMDS) derivative with the DMDS derivative of an authentic standard. A yeast culture was split into 12 aliquots (to not change any parameters in the developed procedure). The cells were pelleted, which yielded 63 mg cells (ccw) on average (755 mg from 18 mL culture). The pellets were subjected to base methanolysis as described above. However, after acidification the samples were combined in a 50 mL Falcon tube. The combined sample was extracted two times with 10 mL chloroform. The mixture was centrifuged 10 min at 3000 rpm to achieve a better phase separation. The combined organic phases, which were combined in a new 50 mL Falcon and were washed consecutively with 10 mL brine and 10 mL water. The organic phase was dried with anhydrous sodium sulfate and concentrated in vacuo. The concentrated oil was dissolved in 1.5 mL chloroform and transferred to a 1.5 mL plastic tube. The chloroform was evaporated at 90° C. The remaining sample was the dissolved in 50 μL methyl tert-butyl ether (MTBE). The 50 μL were split into 1, 5, 10 and 20 μL and transferred into GC-vials without insert. To each vial 200 μL DMDS (dimethyl disulfide) and 50 μL MTBE (containing 60 mg/mL iodine) were added. After the mixture was heated 48 h at 50° C., excess iodine was removed by the addition of 100 μL saturated sodium thiosulfate solution. The samples were transferred to plastic vials and extracted to times with 500 μL dichloromethane. The combined organic phases were transferred to a new 1.5 mL plastic vial and evaporated at 90° C. The samples were taken up in 50 μL DCM and transferred to a GC-vial. The sample was analyzed by GC-MS (Table 7) using the method of Hagström et al. (2013) (Hagström, Å. K. et al. A moth pheromone brewery: production of (Z)-11-hexadecenol by heterologous co-expression of two biosynthetic genes from a noctuid moth in a yeast cell factory. Microb. Cell Fact. 12: 125 (2013)).
Background and Rationale
Engineering microbial production of insect fatty alcohols from fatty acids requires the functional expression of a synthetic pathway. One such pathway comprises a transmembrane desaturase, and an alcohol-forming reductase to mediate the conversion of fatty acyl-CoA into regio- and stereospecific unsaturated fatty acyl-CoA, and subsequently into fatty alcohols. A number of genes encoding these enzymes are found in some insects as well as some microalgae. A number of gene variants were screened to identify enzyme activities that allow the creation of pathways capable of high level synthesis of a single or a blend of insect fatty alcohols. Additionally, these enzymes were screened across multiple hosts (Saccharomyces cerevisiae, Candida tropicalis, and Yarrowia lipolytica) in order to find a suitable host for optimum expression of these transmembrane proteins.
Summary of Approach
S. cerevisiae was engineered previously to express select functional transmembrane desaturase variants to allow synthesis of (Z)-11-hexadecenoic acid from palmitic acid. This allowed the identification and rank-ordering of the variants based on their bioconversion performance (see Example 4).
S. cerevisiae was engineered previously to express select functional transmembrane reductase variants to allow synthesis of (Z)-11-hexadecenol (Z11-16OH) from (Z)-11-hexadecenoic acid. This allowed the identification and rank-ordering of the variants based on their bioconversion performance (see Example 3).
Several fatty alcohol pathways comprised of the most active variant desaturases and reductases identified in the previous screens were assembled.
S. cerevisiae W303A and ΔOLE1 were transformed with the pathway constructs. Functionality of the pathway was assessed via the ability of the recombinant yeasts to synthesize Z11-16OH from palmitic acid.
GC-MS analysis was used to identify and quantify metabolites.
Results
The goal was to engineer one or more insect fatty alcohol biosynthetic pathways in S. cerevisiae. Previously, the functional expression of several transmembrane desaturases of insect origin in S. cerevisiae was demonstrated (see Example 4). Briefly, heterologous desaturase expression was enabled by designing an expression cassette which consists of an OLE1 promoter region, an N-terminal leader sequence encoding the first 27 amino acids of S. cerevisiae OLE1, and a terminator region of VPS13. Screening for active desaturases was done by using two approaches. First, active desaturases were screened for their ability to rescue ΔOLE1 growth without exogenous addition of unsaturated fatty acid (UFA), and second, active desaturases were screened via an in vivo screen for bioconversion of palmitic acid into (Z)-11-hexadecenoic acid. These screening strategies allowed the identification of several active variants, and the rank ordering of their relative activity. Based on these screening results, desaturases from Trichoplusia ni (TN_desat) and S. cerevisiae (SC_desat) were selected for combinatorial expression in fatty alcohol pathways. S. cerevisiae desaturase is known to form palmitoleic acid and oleic acid.
The functional expression of several transmembrane alcohol forming reductases of insect origin in S. cerevisiae had also been previously demonstrated (see Example 3). An expression cassette comprising the GAL1 promoter and CYC terminator was used to enable the functional expression of the reductases in S. cerevisiae. Screening several reductases via in vivo bioconversion of (Z)-11-hexadecenoic acid into Z11-160H allowed the identification of active variants and rank ordering of their relative activity. Based on this screen, reductases from Helicoverpa armigera (HA_reduc), and Spodoptera littoralis (SL_reduc) were chosen for assembly of the fatty alcohol pathways.
Combinatorial assembly created four fatty alcohol pathways, i.e. TN_desat-HA_reduc, TN_desat-SL_reduc, SC_desat-HA_reduc, and SC_desat-SL_reduc. Pathways with SC_desat served as negative control for insect Z11-160H synthesis. S. cerevisiae ΔOLE1 and W303A were transformed with constructs harboring these pathways, and transformants that grew on CM-Ura with 2% glucose and coated with palmitoleic acid were isolated. To test for fatty alcohol production, individual clones were inoculated into CM-Ura medium containing 2% glucose, 1% raffinose, 2% galactose. 300 mg/L palmitic acid, and 360 mg/L palmitoleic acid were added as bioconversion substrates. Bioconversion using palmitic acid without palmitoleic was also tested. Post ˜96 h-cultivation in the presence of palmitic and palmitoleic acid, culture broth analysis revealed synthesis of Z11-9OH as a major C16 alcohol product at ˜0.2 mg/L, and ˜0.3 mg/L in cultivation of ΔOLE1 strains harboring SC_desat-HA_reduc, and TN_desat-HA_reduc, respectively (
The bioconversion of palmitic acid was also tested alone (without exogenous addition of palmitoleic acid) by ΔOLE1 strains expressing TN_desat-HA_reduc and TN_desat-SL_reduc (
OLE1 deletion impairs growth. Therefore, pathway expression was also explored in W303A, a host with intact OLE1 allele. However, despite growth improvement, pathway expression in this host resulted in more than two-fold reduction of Z11-16OH titers. This result was likely due to the repression of OLE1 promoter (which drove heterologous desaturase expression) by endogenous unsaturated fatty acyl:CoAs, the products of OLE1. The S. cerevisiae OLE1 promoter has been previously characterized with structural regions found to be positively and negatively regulated by saturated and unsaturated fatty acid, respectively (Choi, J-Y. et al. Regulatory Elements That Control Transcription Activation and Unsaturated Fatty Acid-mediated Repression of the Saccharomyces cerevisiae OLE1 Gene. J. Biol. Chem. 271: 3581-3589 (1996)). In addition to cis-transcriptional regulation, unsaturated fatty acids also interact with OLE1 promoter elements to regulate mRNA stability (Gonzales, C. I. et al. Fatty acid-responsive control of mRNA stability. Unsaturated fatty acid-induced degradation of the Saccharomyces OLE1 transcript. J. Biol. Chem. 271: 25801-25809 (1996)). Due to this inherent complexity of the OLE1 promoter, the utilization of unregulated orthogonal promoters, such as the OLE1 promoter from S. kluyveri (Kajiwara, S. Molecular cloning and characterization of the v9 fatty acid desaturase gene and its promoter region from Saccharomyces kluyveri. FEMS Yeast. Res. 2: 333-339 (2002)) to drive insect desaturase expression can be explored to enhance fatty alcohol production.
In summary, functional expression of synthetic pheromone pathway variants in S. cerevisiae ΔOLE1 resulted in the synthesis of Z11-16OH and Z9-16OH from palm oil fatty acids (palmitic acid and palmitoleic acid) up to approximately 0.2 mg/L and 0.3 mg/L, respectively.
The engineered pathway that resulted in the highest fatty alcohols is comprised of T. ni desaturase and H. armigera reductase.
Accumulation of (Z)-11-hexadecenoic acid, an intermediate of the pathway, was also observed in strains that produced Z11-16OH.
No Z11-16OH was produced and only trace Z9-16OH was detected in the negative control strain (harboring vector only).
The regio- and stereochemistry of the biologically produced Z11-16OH were confirmed by comparing the retention time and fragmentation pattern to the authentic standard compound via GC-MS.
Conclusions
The engineering of Baker's yeast for synthesis of Z11-16OH and Z9-16OH, fatty alcohol precursors of insect pheromones, was demonstrated.
Fatty alcohol production varies depending on the selection of the desaturase and reductase variants.
Accumulation of (Z)-11-hexadecenoic acid suggested the possibility of further fatty alcohol improvement by increasing the performance of alcohol forming reductase. However, it is also possible that detection of (Z)-11-hexadecenoic acid was due to its incorporation as phospholipid into any membrane other than the endoplasmic reticulum membrane (such as mitochondrial membranes, peroxisome, nuclear envelope, etc), therefore inaccessible to alcohol forming reductase (presumably translocated into the endoplasmic reticulum) which must utilize (Z)-11-hexadecenoic acid in its CoA thioester moiety as its substrate.
Culture conditions can be explored to increase fatty alcohol titers. The T. ni desaturase can be replaced in the pathway by A. transitella desaturase, another variant that also showed high activity and rescued ΔOLE1 growth faster than T. ni desaturase. The synthetic pathway can be imported into Candida tropicalis and Yarrowia lipolytica, which are yeasts with high adhesion property to hydrophobic substrates such as palmitic and palmitoleic acid. By increasing substrate accessibility to the microbial production platform, it is foreseeable that product titer and yield can be improved.
Materials & Methods
Strain Construction and Functional Expression Assay
S. cerevisiae ΔOLE1 (MATA OLE1::LEU2 ura3-52 his4), and W303A (MATA ura3-1 trp1-1 leu2-3_112 his3-11_15 ade2-1 can1-100) were used as expression hosts. Modular design allows combinatorial pathway assembly utilizing BamHI and XhoI to excise reductase synthons (see Example 3) and subcloning into plasmids containing pOLE1-desaturase constructs (see Example 4). Competent yeasts were transformed with pathway constructs and plated on CM-Ura glucose agar plate (Teknova). In the case of ΔOLE1 transformation, colony plating utilized 20 mM CM-Ura glucose agar plates that were coated with 100 μL CM-Ura glucose medium containing 1% tergitol and 3 μL palmitoleic acid.
To assess functional expression, transformants were inoculated in ˜20 mL CM-Ura liquid medium containing 6.7 g/L of YNB, 2% glucose, 1% raffinose, and 2% galactose. Fatty acid substrates, i.e. palmitic acid (in ethanol), was added at a final concentration of 300 mg/L. Palmitoleic acid was added at a final concentration of 360 mg/L. Bioconversion assay proceeded for 96 h at 28° C. prior to GC-MS analysis.
Metabolite Extraction and GC-MS Detection
Fatty acid analysis was as described in Example 4, except that instead of extracting the sample two times, the sample was only extracted once with chloroform containing 1 mM methyl heptadecanoate (C17:0Me). Fatty alcohol analysis was as described in Example 3, except that instead of hexane (containing tetradecanedioic acid), chloroform (containing 1 mM methyl heptadecanoate) was used. The extraction time was reduced from 1 h to 20 s. Afterwards the samples were collected in a 1.8 mL GC vial and not in a 1.5 mL plastic tube. The mass spectrometer was used in SIM mode (m/z 208, 297.3 and 387.3).
Example 6 Expression of Transmembrane Desaturases in Candida tropicalisBackground and Rationale
Engineering microbial production of insect fatty alcohols from fatty acids requires the functional expression of a synthetic pathway. One such pathway comprises a transmembrane desaturase, and an alcohol-forming reductase to mediate the conversion of fatty acyl-CoA into regio- and stereospecific unsaturated fatty acyl-CoA, and subsequently into fatty alcohols. A number of genes encoding these enzymes are found in some insects as well as some microalgae. A number of gene variants were screened to identify enzyme activities that allow the creation of pathways capable of high level synthesis of a single or a blend of insect fatty alcohols. Additionally, these enzymes can be screened across multiple hosts (Saccharomyces cerevisiae, Candida tropicalis, and Yarrowia lipolytica) to optimize the search toward finding a suitable host for optimum expression of these transmembrane proteins.
Summary of Approach
A small set of desaturases (insect origin: Agrotis segetum, Amyelois transitella, Helicoverpa zea, Trichoplusia ni, Ostrinia furnacalis, and Lampronia capitella and marine diatom: Thalassiosira pseudonana) were selected as a test case to explore and establish functional expression assays, metabolite extraction methods, and analytical chemistry.
Successful integration and functional expression of mCherry control from pXICL expression cassette in SPV053 were confirmed.
A recombinant desaturase library using the same pXICL vector in SPV053 background was integrated (
Functionality of the desaturase was validated via an in vivo bioconversion of hexadecanoic acid (palmitic acid) into (Z)-11-hexadecenoic acid (palmitvaccenic acid).
GC-FID and GC-MS analyses were used to identify and quantify metabolites.
Results
Library Construction
This study focused on the screening for transmembrane desaturase variants in C. tropicalis (SPV053). Five insect desaturases with reported Z11 desaturase activity on palmitoyl-CoA (C16:0) (SEQ ID NOs: 16-19, 23) and three insect desaturases with reported Z9 desaturase activity (SEQ ID NOs: 20-22) were included in the screen. One variant, the Z11 desaturase from A. segetum (SEQ ID NO: 16), was also cloned with 27 amino acids of the Candida albicans OLE1 N-terminus fused upstream of the insect sequence (
Transformation efficiencies of linearized plasmids into SPV053 varied greatly across constructs. Despite low efficiencies, at least 3 clonal isolates were identified for each variant (Tables 8 and 9). It had been hypothesized that larger colonies on transformation plates were more likely to be positive integrants because the presence of the Zeocin resistance marker should increase growth rate under Zeocin selection. Analysis of the screening results suggested that the number of large colonies is not correlated to transformation efficiency. Instead total colony (small and large) count correlated best with observed efficiency (
Functional Expression Assay
Functional expression of the heterologous desaturases was characterized by a series of in vivo bioconversion experiments. C. tropicalis SPV053 derived stains expressing insect desaturases were cultured in rich (YPD) or defined (CM glucose) media supplemented with ethanol (for induction) and saturated acid substrates (palmitic acid, methyl palmitate, methyl myristate). Small scale (2 ml) cultures were cultivated for a total of 72 hours in 24 deep well plates with substrate added after the initial 24 hours.
The first screen examined multiple bioconversion media with supplementation of a palmitic acid substrate. Two functional palmitoyl-CoA (Z)-11 desaturases were identified by fatty acid methyl ester (FAME) analysis of the cellular lipid content. Strains expressing A. transitella or H. zea Z11 desaturases (SPV0305-SPV0310) produced a fatty acid species not observed in the mCherry control strains (SPV0302-SPV0304) which eluted with the (Z)-11-hexadecenoic acid standard (
The bioconversion assay was scaled-up to 20 ml in shake flasks in order to generate enough biomass for additional characterization of the putative (Z)-11-hexadecenoic acid species. While the observed species eluted with the (Z)-11-hexadecenoic acid standard and independently of the (Z)-9-hexadecenoic acid standard, it was possible that a different fatty acid isomer (e.g. (E)-9-hexadecenoic acid) could have a similar retention time to (Z)-11-hexadecenoic acid. As different stereoisomers elute differently on the DB-23 the occurrence of (E)-11-hexadecenoic could be excluded. Final confirmation of (Z)-11-hexadecenoic acid production was completed by using mass spectroscopy detection of DMDS derivatized fatty acids to confirm the 11-regioselectivity. Using this derivatization technique (Z)-11 and (E)-11 isomers could in principle also be resolved. The fragmentation pattern of experimental samples could be matched to the (Z)-11-hexadecenoic acid standard (
Finally, methyl myristate (C14:0) was tested as substrate for the entire desaturase library. A non-native fatty acid species which elutes between myristate (C14:0) and (Z)-9-tetradecenoic acid (Z9-C14:1) was observed in strains expressing either A. transitella or H. zea Z11 desaturases (
In summary, two desaturases from Helicoverpa zea (AAF81787) and from Amyelois transitella (JX964774), were expressed in SPV053 and conferred synthesis of (Z)-11-hexadecenoic acid from either endogenously produced or supplemented palmitic acid.
Functional expression of H. zea and A. transitella desaturases in C. tropicalis SPV053 was confirmed using an in vivo bioconversion assay in both rich (YPD) and defined (CM glucose) media. The active desaturases generated intracellular (Z)-11-hexadecenoic acid which was not observed in mCherry expressing control strains. C16-fatty acid composition of SPV053 expressing H. zea desaturase is approximately 50.0% hexadecanoic acid, 30.91% (Z)-9-hexadecenoic acid and 19.1% (Z)-11-hexadeceneoic acid. With palmitic acid supplementation the composition is 58.1% hexadecanoic acid, 17.5% (Z)-9-hexadecenoic acid and 24.4% (Z)-11-hexadeceneoic acid. The C16-fatty acid composition of SPV053 expressing A. transitella desaturase is 55.5% hexadecanoic acid, 14.5% (Z)-9-hexadecenoic acid and 30.0% (Z)-11-hexadeceneoic acid. In comparison, SPV053 expressing mCherry produced a C16-fatty acid composition of approximately 72.9% hexadecanoic acid, 27.1% (Z)-9-hexadecenoic acid and no (Z)-11-hexadeceneoic acid. (Z)-11-hexadecenoic acid was produced at approximately 5.5 mg/L in both strains expressing functional Z11 desaturases.
No (Z)-11-hexadecenoic acid was observed in strains harboring T. ni, T. pseudonana, or A. segetum desaturase.
No difference in fatty acid composition was observed for strains expressing Z9 insect desaturases from H. zea, O. furnacalis, or L. capitella.
The regio- and stereoisomer of the biologically produced (Z)-11-hexadecenoic acid were confirmed by comparing the retention time and fragmentation pattern of the authentic standard compound via GC-MS after DMDS derivatization.
Bioconversions of SPV053 expressing A. transitella and H. zea desaturases with supplementation of methyl myristate produced an unidentified metabolite not observed in the mCherry expressing negative control strain. The GC retention time of this metabolite is found between myristate (C14:0) and (Z)-9-tetradecenoic acid.
Conclusions
Functional expression of transmembrane desaturase of insect origin in C. tropicalis SPV053 has been achieved.
The active desaturases identified via screening in C. tropicalis also complemented OLE1 function when expressed in S. cerevisiae ΔOLE1 (See Example 4).
An in vivo assay can be used to assay desaturase activity in C. tropicalis for non-native fatty acid isomers (e.g. (Z)-11-hexadecenoic acid). Enhanced ratios of non-native fatty acids can be produced with supplementation of saturated acid substrates such as palmitic acid or methyl myristate.
Functional expression and/or activity of insect desaturases varies widely in C. tropicalis SPV053 depending on sequence origin. Similar to results observed in the S. cerevisiae screen (See Example 4), A. segetum and T. pseudonana variants did not produce detectable (Z)-11-hexadecenoic acid. Interestingly, T. ni desaturase also failed to produce detectable (Z)-11-hexadecenoic acid under assay conditions. Unlike in the S. cerevisiae assay, the T. ni expression construct did not include a chimeric OLE1 leader sequence.
The inclusion of the C. albicans OLE1 leader sequence on the functional H. zea variant and non-functional T. ni variant can be tested.
The functional expression of additional desaturase variants to identify C14-specific desaturases can be explored.
Expression of functional desaturase with reductase variants can be done and subsequent screen for unsaturated fatty alcohol production can be performed.
Materials & Methods
Strain Construction
A conservative approach was used for recoding of genes. Native sequences were unaltered except for replacement of CTG leucine codons with TTA. All genes were cloned into pPV0053 using NcoI and NotI restriction sites by Genscript. After transformation into E. coli NEB10β, plasmids were miniprepped using the Zyppy Plasmid Miniprep Kit (Zymo Research, Irvine, CA). Plasmids were linearized by digestion with BsiWI (New England Biolabs, Ipswich, MA) before transformation into SPV053. After digestion, DNA was isolated using Clean and Concentrator Kit (Zymo Research, Irvine, CA). Approximately 1 μg of DNA was transformed by electroporation. Instead of incubation with TE+100 mM lithium acetate+DTT, cells were incubated in only TE+100 mM lithium acetate for 2 hours. Positive integrants were found to be site-specific and genotyping was conducted by check PCR. A two-stage approach was adopted for further screening of low efficiency transformations. Approximately 60 colonies were re-patched on YPD+300 μg/ml Zeocin and grown overnight. The subset of patches which grew quickly (dense growth within 24 hours) were screened by colony PCR. The vast majority of rapid growing patches were identified as positive integrants.
Functional Expression Assay
Palmitic Acid Supplementation in YPD and CM Glucose
Positive isolates were re-patched onto YPD+300 μg/ml Zeocin and grown overnight and then stored at 4° C. Strains were inoculated from patch plates into 2 ml of YPD in 24 deep well plates (square well, pyramid bottom). Three positive clones were inoculated for each desaturase variant and the mCherry expressing control strain. Deep well plates were incubated at 30° C., 1000 rpm, and 80% humidity in the Infors HT Multitron Pro plate shaker for 24 hrs. After 24 hrs of incubation, cultures were split into equal 1 ml volumes to make two sets of identical plates. Both sets of plates were pelleted by centrifugation at 500×g. One set of plates was resuspended in 2 ml of YPD+0.3% (v/v) ethanol and the second set was resuspended in 2 ml of CM glucose+0.3% ethanol. Ethanol was added at this stage to induce recombinant enzyme expression from the ICL promoter. Cultures were incubated for another 24 hours under the same conditions before 300 mg/L palmitic acid was added to cultures from a 90 g/L stock solution in ethanol. The result was the addition of a fresh 0.3% ethanol in conjunction with the palmitic acid. A subset of strains was also cultured without palmitic acid addition. These cultures had 0.3% ethanol added instead. All cultures were incubated for an additional 24 hrs before a final addition of 0.3% ethanol. After another 24 hr period of incubation, 1.5 ml of each culture was harvested in 1.7 ml microcentrifuge tubes and pelleted. Supernatant was saved in fresh tubes and pellets were processed as described below. A subset of supernatant samples was also extracted to look for free acid in the extracellular medium.
Repeated Screening with Alternate Substrates
The mCherry control and confirmed positive variants were rescreened using both palmitic acid and methyl palmitate as substrates. The culturing was conducted as described above with equimolar (1.17 mM) amounts of substrate added from ethanol stock solutions (methyl palmitate 94 g/L stock, 313 mg/L final concentration). The same protocol was also repeated with the full panel of strains using an 84 g/L stock of methyl myristate (C14:0). The final concentration of substrate was again 1.17 mM.
Confirmation of (Z)-11-hexadecenoic Acid Isomer
The in vivo bioconversion assay was scaled up for confirmation of (Z)-11-hexadecenoic acid synthesis. 2 ml YPD seed cultures of strains SPV0302, SPV0303, and SPV0304 (mCherry), SPV0304, SPV0305, and SPV0306 (A. transitella Z11 desaturase), and SPV0307, SPV0308, and SPV0309 (H. zea Z11 desaturase) were grown overnight at 30° C., 1000 rpm, 80% humidity in the Infors HT Multitron plate shaker. 200 μl of overnight culture from each of the three clonal isolates was pooled and inoculated into a single 125 ml baffled flask containing 20 ml YPD. The resulting three flasks were grown for 24 hrs at 30° C. and 250 rpm (Infors Flask shaker). Cultures were pelleted by centrifugation at 500×g and resuspended in 20 ml of YPD+0.3% (v/v) ethanol and returned to 125 ml baffled shake flasks. Cultures were incubated for an additional 24 hours before addition of 300 mg/L palmitic acid in a 90 g/L stock in ethanol (221 μl per flask). After 24 hours of incubation another 0.3% (v/v) ethanol (221 μl) was added to each flask for sustained induction. Flasks were incubated for an additional 24 hours before cells were harvested for FAME analysis and DMDS derivatization.
Metabolite Extraction and GC-MS Detection
Total lipid composition as well as the (Z)-11-hexadecenoic acid quantification was based on modified procedures by Moss et al. (1982) and Yousuf et al (2010). The pelleted cells (in 1.5 mL plastic tubes), usually about 10 mg to 80 mg, were resuspended in methanol containing 5% (w/w) of sodium hydroxide. The alkaline cell suspension was transferred into a 1.8 mL screw-cap GC-vial. The mixture was heated for 1 h in the heat block at 90° C. Prior to acidification with 400 2.5 N HCl the vial was allowed to cool to room temperature. 500 μL chloroform containing 1 mM heptadecanoic were added and the mixture was shaken vigorously, then both aqueous and organic phase were transferred into a 1.5 mL plastic tube. The mixture was centrifuged at 13,000 rpm, afterwards 450 μL of the organic phase were transferred into a new 1.5 mL plastic tube. The aqueous phase was extracted a second time with 500 μL chloroform, this time without heptadecanoic acid. The combined organic phases were evaporated at 90° C. After cooling to room temperature, residual fatty acid methyl esters and free fatty acids were dissolved and derivatized in methanol containing 0.2 M TMSH (trimethylsulfonium hydroxide).
The regioselectivity of biologically produced (Z)-11-hexadecenoic acid was determined by comparing the fragmentation patterns of the dimethyl disulfide (DMDS) derivative with the DMDS derivative of an authentic standard. A yeast culture was split into 12 aliquots (to not change any parameters in the developed procedure). The cells were pelleted, which yielded 63 mg cells (ccw) on average (755 mg from 18 mL culture). The pellets were subjected to base methanolysis as described above. However, after acidification the samples were combined in a 50 mL falcon tube. The combined sample was extracted two times with 10 mL chloroform. The mixture was centrifuged 10 min at 3000 rpm to achieve a better phase separation. The combined organic phases were combined in a new 50 mL falcon and were washed consecutively with 10 mL brine and 10 mL water. The organic phase was dried with anhydrous sodium sulfate and concentrated in vacuo. The concentrated oil was dissolved in 1.5 mL chloroform and transferred to a 1.5 mL plastic tube. The chloroform was evaporated at 90° C. The remaining sample was the dissolved in 50 μL methyl tent-butyl ether (MTBE). The 50 μL were split into 1, 5, 10 and 20 μL and transferred into GC-vials without insert. To each vial 200 μL DMDS (dimethyl disulfide) and 50 μL MTBE (containing 60 mg/mL iodine) were added. After the mixture was heated 48 h at 50° C., excess iodine was removed by the addition of 100 μL saturated sodium thiosulfate solution; however, due to excessive formation of detergents from the Candida strain, the layer did not mix properly. The samples were therefore diluted in a 15 mL falcon tube to a final sample composition of 200 μL, 3.55 mL MTBE (containing iodine and analyte), 500 μL dichloromethane, 1.5 mL water and 1 mL ethanol. The organic phase was evaporated stepwise at 85° C. in a 1.8 mL glass vial. The samples were taken up in 500 μL dichloromethane and the sample was analyzed by GC-MS using the method of Hagström et al. (2013) as in Example 4.
Example 7 Expression of Transmembrane Desaturases in Yarrowia lipolyticaBackground and Rationale
Engineering microbial production of insect fatty alcohols from fatty acids requires the functional expression of a synthetic pathway. One such pathway comprises a transmembrane desaturase, and an alcohol-forming reductase to mediate the conversion of fatty acyl-CoA into regio- and stereospecific unsaturated fatty acyl-CoA, and subsequently into fatty alcohols. A number of genes encoding these enzymes are found in some insects as well as some microalgae. Alternatively, regio- and stereospecific desaturases can be used to produce a microbial oil rich in fatty acid precursors. The microbial oil can then be derivatized and reduced to active ingredients. A number of gene variants were screened to identify enzyme activities that allow the creation of pathways capable of high level synthesis of a single or a blend of insect fatty acids and alcohols. Additionally, these enzymes were screened across multiple hosts (Saccharomyces cerevisiae, Candida viswanathii (tropicalis), and Yarrowia lipolytica) to optimize the search toward finding a suitable host for optimum expression of these transmembrane proteins.
Initial screening of desaturases in S. cerevisiae and C. viswanathii (tropicalis) identified three active Z11-C16:1 desaturase variants from Amyelois transitella, Helicoverpa zea, and Trichoplusia ni. The S. cerevisiae screening used coding sequences with an N-terminal leader sequence of the S. cerevisiae Ole1p Z9 desaturase fused to the full length insect Z11 desaturase sequence. This strategy has been used previously in the scientific literature to express eukaryotic desaturases in S. cerevisiae. All three of the above desaturases displayed Z11 desaturase activity with the Ole1p leader fusion when expressed in a OLE1 deletion background. An analogous design with a C. albicans Ole1p leader sequence was used with the Z11 desaturase from H. zea. While active, this Ole1p-H. zea desaturase fusion did not significantly increase Z11-hexadecenoic acid titer. Additionally, a conservatively optimized A. transitella Z11 desaturase was active in both S. cerevisiae and C. viswanathii. The following study focused on testing the functional expression of the H. zea, T. ni, and A. transitella Z11 desaturases in two different Y. lipolytica strains, SPV140 and SPV300. Both native and Homo sapiens codon optimized sequences were used for the H. zea and T. ni desaturases while only the native sequence was used for A. transitella. Finally, the N-terminus of the Y. lipolytica Ole1p Z9 stearoyl-CoA desaturase aligns more closely with insect desaturases than the N-terminus of Ole1p from either S. cerevisiae or C. albicans. Based on this alignment two additional desaturase versions were created. A putative leader sequence was swapped from the Y. lipolytica Ole1p onto the T. ni and H. zea desaturases.
Summary of Approach
A focused library of Z11 desaturases (insect origin: Amyelois transitella, Helicoverpa zea, Trichoplusia ni), which had observed activity in either S. cerevisiae or C. viswanathii were cloned into a double crossover cassette targeting the XPR2 locus with a URA3 selection marker. Protein coding sequences use either the native insect sequence (SEQ ID NOs: 24, 25), Homo sapiens optimized coding sequence (SEQ ID NOs: 26, 27), or the Homo sapiens optimized sequence with the N-terminal 84 bases (H. zea, SEQ ID NO: 29) or 81 bases (T. ni, SEQ ID NO: 28) swapped for the N-terminal 96 bases of the Y. lipolytica OLE1 (YALI0005951) gene. Unlike in the S. cerevisiae and C. viswanathii screens, the leader sequence chimeras test a direct swap of leader sequences instead of concatenating a host leader sequence to the N-terminus of the full length desaturase coding sequence. Only the native coding sequence was used for the A. transitella desaturase (SEQ ID NO: 30).
Each of the 7 desaturase constructs was transformed into SPV140 (PO1 f) and SPV300 (H222 ΔP ΔA ΔF ΔURA3) and site-specific integrants were confirmed.
Desaturase activity was tested via an in vivo bioconversion of hexadecanoic acid (palmitic acid) into (Z)-11-hexadecenoic acid (palmitvaccenic acid) in YPD medium.
GC-FID analyses were used to identify and quantify metabolites.
Results
Strain Construction
Desaturase variants were cloned into the pPV101 vector which contains a Y. lipolytica expression cassette targeting integration into the XPR2 locus (YALI0F31889g).
The T. ni and H. zea desaturases were each synthesized with the native insect sequence (SEQ ID NOs: 24, 25), full length insect sequence codon optimized for Homo sapiens (SEQ ID NOs: 26, 27), or with the putative leader sequence replaced by the leader sequence from Y. lipolytica OLE1 desaturase (SEQ ID NOs: 28, 29). The A. transitella desaturase was also synthesized using the native insect coding sequence (SEQ ID NO: 30). All seven desaturase variants were transformed into SPV140. Based on previous activity results, only the H. zea and A. transitella desaturase variants were transformed into SPV300.
Functional Expression Assay
Functional activity was assessed by a modification of the protocol used for transmembrane desaturase expression in C. viswanathii SPV053 (See Example 6). Briefly, Y. lipolytica SPV140 and SPV300 derived stains expressing insect desaturases were cultured in rich (YPD) to generate biomass. Using the YPD generated biomass, small scale (2 ml) cultures were cultivated with palmitic acid for a total of 48 hours in 24 deep well plates (See Materials & Methods for detail).
In the initial screen of T. ni, H. zea, and A. transitella variants, only H. zea desaturase variants that were codon optimized for Homo sapiens produced detectable Z11-hexadecenoic acid (
A follow up experiment was conducted comparing active variants in the SPV140 background to SPV300 derived desaturase strains. The parent SPV300 and SPV140 expressing hrGFP were used as negative controls. The same bioconversion assay protocol was used. As in SPV140, only H. sapiens optimized variants produced detectable activity (
In summary, only the H. zea Z11 desaturase variants with Homo sapiens codon optimization produced detectable Z11-hexadecenoic acid. Under the current assay condition, marginally higher titers were observed in the SPV140 background over SPV300. Table 11 summarizes the Z11-hexadecenoic acid titers.
In SPV300, one non-site-specific integrant of pPV200 (Y. lipolytica OLE1-H. zea Z11 desaturase with Homo sapiens codon optimization) was tested. This integrant did not produce detectable Z11-hexadecenoic acid, while the two site-specific integrants produced 55±1 mg/L.
No major hydroxy or diacid peaks were observed from pellets of SPV140 or SPV300 derived strains, and deletion of β-oxidation/ω-oxidation genes in SPV300 did not increase Z11-hexadecenoic acid accumulation under the current assay condition (relatively low substrate concentration, rich medium).
Conclusions
The H. zea Z11 desaturase is active and confers production of ˜100 mg/L Z11-hexadecenoic acid, from ˜500 mg/L palmitic acid substrate. The functional expression was demonstrated across three positive integrants and replicate experiments in a 24 well plate assay.
H. zea desaturase required codon optimization (Homo sapiens or potentially Y. lipolytica) for activity in Y. lipolytica.
The T. ni Z11 desaturase, while active in S. cerevisiae, does not produce detectable Z11-hexadecenoic acid in Y. lipolytica.
The reproducibility of the assay for Y. lipolytica strains can be confirmed starting from glycerol stock.
A. transitella desaturase can be codon optimized for expression in Y. lipolytica.
Since Y. lipolytica is a candidate production host, additional copies of active desaturases can be integrated in Y. lipolytica, culture conditions to improve bioconversion can be identified, and substrate conversion can be quantified.
Materials & Methods
Strain Construction
All desaturase genes were synthesized (Genscript). Either native sequences or Homo sapiens codon optimization was used. Synthesized genes were subcloned into pPV101. Plasmids were transformed and prepped from E. coli EPI400 using the Zyppy Plasmid Miniprep Kit (Zymo Research, Irvine, CA). Approximately ˜1-2 μg of linearized DNA was transformed using Frozen-EZ Yeast Transformation II Kit (Zymo Research, Irvine, CA). The entire transformation mixture was plated on CM glucose-ura agar plates. Positive integrants were found to be site-specific and genotyping was conducted by check PCR.
Functional Expression Assay
Palmitic Acid Supplementation in YPD
Positive isolates were re-patched onto YPD, grown overnight, and then stored at 4° C. Strains were inoculated from patch plates into 2 ml of YPD in 24 deep well plates (square well, pyramid bottom). Three positive clones were inoculated for each desaturase variant. Three isolates of pPV101 in SPV140 and the parent SPV300 were used as negative controls. Deep well plates were incubated at 28° C. and 250 rpm in the Infors Multitron refrigerated flask shaker for 24 hrs. After 24 hrs of incubation, a 1 ml volume of each culture was pelleted by centrifugation at 500×g. Each pellet was resuspended in 2 ml of YPD. 500 mg/L palmitic acid was added to cultures from a 90 g/L stock solution in ethanol. The result was the addition of 0.5% ethanol with the palmitic acid substrate. All cultures were incubated for 48 hours before endpoint sampling. Final cell densities were measured with the Tecan Infinite 200pro plate reader. 0.75 or 0.8 ml of each culture was harvested in 1.7 ml microcentrifuge tubes and pelleted. Supernatant was removed and pellets were processed as described below.
Metabolite Extraction and GC-FID Analysis
Total lipid composition as well as the (Z)-11-hexadecenoic acid quantification was based on modified procedures by Moss et al. (1982) and Yousuf et al (2010). The pelleted cells (in 1.5 mL plastic tubes), usually about 10 mg to 80 mg, were resuspended in methanol containing 5% (w/w) of sodium hydroxide. The alkaline cell suspension was transferred into a 1.8 mL crimp vial. The mixture was heated for 1 h in the heat block at 90° C. Prior to acidification with 400 2.5 N HCl the vial was allowed to cool to room temperature. 500 μL chloroform containing 1 mM methyl heptadecanoate were added and the mixture was shaken vigorously, then both aqueous and organic phase were transferred into a 1.5 mL plastic tube. The mixture was centrifuged at 13,000 rpm, afterwards 450 μL of the organic phase were transferred into a GC vial. For the analysis of lipids and the quantification of fatty acids 50 μL of 0.2 M TMSH (trimethylsulfonium hydroxide in methanol) was added and the sample analyzed by GC-FID.
Example 8 Candida viswanathii (tropicalis) as a Production Platform for Insect Fatty Alcohol SynthesisBackground and Rationale
Variants of insect transmembrane desaturases and reductases were previously screened and rank-ordered based on their functional expression in either Candida viswanathii or Saccharomyces cerevisiae (see Examples 3, 4 and 6). Helicoverpa zea desaturase and Helicoverpa armigera reductase were selected to assemble a synthetic insect fatty alcohol pathway in C. viswanathii. Simultaneous expression of codon optimized H. zea desaturase under Candida isocitrate lyase (ICL) promoter, and codon optimized H. armigera reductase under Candida transcription elongation factor (TEF) promoter was achieved via genomic integration of the full fatty alcohol pathway. Accumulation of Z11-16OH was achieved in cultures of the recombinant strain (SPV0490) using simple carbon sources and palmitic acid.
Summary of Approach
Integration plasmids were designed containing a functional Helicoverpa zea desaturase (See Example 6) paired with a Helicoverpa armigera reductase driven by a putatively constitutive C. tropicalis promoter (pTEF).
Functionality of the full pathway was assessed via an in vivo bioconversion of hexadecanoic acid (palmitic acid) into Z11-16OH.
GC-FID and GC-MS analyses were used to identify and quantify metabolites.
Results
Accumulation of Z11-16OH was detected in cultures of Candida engineered to express H. zea desaturase under an ICL promoter and H. armigera reductase under a TEF promoter (Table 12 and
Materials & Methods
Strain Construction
The integration plasmid (ppV0228) was designed to contain two expression cassettes. The first cassette contains H. zea codon-optimized desaturase (SEQ ID NO: 31) that was driven by the C. viswanathii ICL promoter (SEQ ID NO: 33). The second cassette contains codon-optimized H. armigera reductase (SEQ ID NO: 32) driven by the C. tropicalis TEF promoter (SEQ ID NO: 34). Gene expression in the ICL promoter cassette is terminated by the ICL terminator sequence (SEQ ID NO: 35). Gene expression in the TEF promoter cassette is terminated by the TEF terminator sequence (SEQ ID NO: 36). A conservative approach was used for recoding of genes. Native gene sequences were unaltered except for replacement of CTG leucine codons with TTA. After transformation into E. coli NEB100, plasmids were miniprepped using the Zyppy Plasmid Miniprep Kit (Zymo Research, Irvine, CA). Plasmids were linearized by digestion with BsiWI (New England Biolabs, Ipswich, MA) before transformation into SPV053. After digestion, DNA was isolated using Clean and Concentrator Kit (Zymo Research, Irvine, CA). Approximately 3-5 μg of DNA was transformed by electroporation. Positive integrants were found to be site-specific and genotyping was conducted by check PCR. A two-stage approach was adopted for further screening of low efficiency transformations. Approximately 100 colonies were re-patched on YPD+250 μg/ml Zeocin and grown overnight. The subset of patches which grew quickly (dense growth within 24 hours) were screened by colony PCR.
Functional Expression Assay
Palmitic Acid Supplementation in YPD
Positive isolates were re-patched onto YPD+300 μg/ml Zeocin, grown overnight and then stored at 4° C. Strains were inoculated from patch plates into 2 ml of YPD in 24 deep well plates (square well, pyramid bottom). Four positive clones were inoculated for each desaturase and reductase variant and three positive clones were inoculated for each desaturase and mCherry expressing control strain. Deep well plates were incubated at 30° C., 1000 rpm, and 80% humidity in the Infors HT Multitron Pro plate shaker for 24 hrs. After 24 hrs of incubation, a 1 ml volume of each culture was pelleted by centrifugation at 500×g. Each pellet was resuspended in 2 ml of YPD+0.3% (v/v) ethanol. Ethanol was added at this stage to induce recombinant enzyme expression from the ICL promoter. Cultures were incubated for another 24 hours under the same conditions before 300 mg/L palmitic acid was added to cultures from a 90 g/L stock solution in ethanol. The result was the addition of a fresh 0.3% ethanol in conjunction with the palmitic acid. All cultures were incubated for an additional 24 hrs before a final addition of 0.3% ethanol. After another 24 hr period of incubation, 1.5 ml of each culture was harvested in 1.7 ml microcentrifuge tubes and pelleted. Supernatant was removed and pellets were processed as described below.
Metabolite Extraction and GC-MS Detection
The pelleted cells (in 1.5 mL plastic tubes), usually about 10 mg to 80 mg, were resuspended in methanol containing 5% (w/w) of sodium hydroxide. The alkaline cell suspension was transferred into a 1.8 mL crimp vial. The mixture was heated for 1 h in a heat block at 90° C. Prior to acidification with 400 μL 2.5 N HCl the vial was allowed to cool to room temperature. 500 μL chloroform containing 1 mM methyl heptadecanoate were added and the mixture was shaken vigorously, then both aqueous and organic phase were transferred into a 1.5 mL plastic tube. The mixture was centrifuged at 13,000 rpm, afterwards 450 μL of the organic phase were transferred into a GC vial. The organic phase was evaporated in a heat block at 90° C. for 30 min. The residue was dissolved in 50 μL N,O-Bis(trimethylsilyl)trifluoroacetamide containing 1% trimethylchlorosilane. Prior to transfer into glass inserts the mixture was heated 5 min at 90° C. The samples were analyzed by GC-MS (Table 13).
Background and Rationale
Yarrowia lipolytica was engineered as a production platform for insect fatty alcohol (Z11-16OH and Z9-16OH) synthesis from palmitic acid.
After individually confirming functional expression of a Z11 desaturase (Example 7) and fatty acyl-CoA reductase (FAR), the full Z11-16OH and Z9-16OH pathways (Bdr) were engineered in Y. lipolytica. For the purpose of improving fatty alcohol titers, cultivations designed for promoting growth vs. for eliciting lipid storage were also explored. A growth condition favors high biomass production, but limits fatty acyl-CoA pool size used by the engineered pathway and directs fatty acyl-CoA intermediates to membrane synthesis. Conversely, a lipid storage condition creates a strong sink for production of fatty acyl-CoAs which is desirable. However, fatty acyl-CoA transport towards lipid bodies creates a strong competition for FAR activity. Under this second scenario, even though Z11-16Acid or Z9-16Acid accumulates in the cell, most of it is inaccessible to the FAR. On the other hand, there may be a continual flux of lipid remobilization under lipid storage conditions which leads to a sustained pool of Z11-16CoA or Z9-16CoA which is available to the FAR.
Summary of Approach
Two biodesaturation-reduction (Bch) pathway variants were tested in the H222 ΔPΔAΔF (SPV300) background. The first combined recombinant expression of Helicoverpa zea Z11 desaturase paired with a Helicoverpa armigera fatty acyl-CoA reductase (FAR amino acid sequence set forth in SEQ ID NO: 41) creating a Z11-16OH synthesis pathway. The second combined native Y. lipolytica Z9 desaturase activity with H. armigera fatty acyl-CoA reductase (FAR) expression creating a Z9-16OH pathway.
Two integration plasmids were constructed to express the H. zea desaturase and the H. armigera FAR. The TEF promoter was used for desaturase expression and the EXP1 (export protein) or the TAL1 (transaldolase) promoter was used for reductase expression.
Successful integration of the Z11-16OH pathway cassette into the H222 ΔPΔAΔF (SPV300) background was confirmed by colony PCR.
Functionality of the full Z11-16OH pathway was assessed via an in vivo bioconversion of 16Acid (palmitic acid) into Z11-16OH (Z-11-hexadecenol).
Functionality of a full Z9-16OH pathway was assessed via an in vivo bioconversion of 16Acid (palmitic acid) using previously constructed SPV471 (H222 ΔPΔAΔF derived) which expresses the H. armigera FAR driven by the TEF promoter.
GC-MS analysis was used to identify and quantify Z9-16OH and Z11-16OH. GC-FID analysis was used to identify and quantify fatty acids.
Summary
Ten isolates expressing the H. zea desaturase (pTEF) and H. armigera reductase (pEXP1) were screened. The in vivo bioconversion assay confirmed Z11-16OH production from all isolates.
Relatively low, detectable Z11-16OH titers (0.26±0.09 mg/L) were observed in a YPD medium supplemented with 10 g/L methyl palmitate. The Z11-16Acid precursor was measured at 220±11 mg/L (across clones 2, 4, 9, 17, 23).
Higher Z11-16OH titers were observed in a semi-defined medium with C:N ratio of ˜80. Across all 10 isolates Z11-16OH was produced at 2.65±0.36 mg/L. The Z11-16Acid precursor titer was 900±30 mg/L. One isolate (SPV578) produced 3.68±0.31 mg/L Z11-16OH (Z11-16Acid 840±14 mg/L).
Nine isolates expressing the H. zea desaturase (pTEF) and H. armigera reductase (pTAL1) were screened. The in vivo bioconversion assay confirmed Z11-16OH production from all isolates.
One isolate (SPV603) produced 6.82±1.11 mg/L Z11-16OH in a semi-defined medium (Z11-16Acid 1.36 g/L).
The previously tested reductase strain, SPV471 (H222 ΔPΔAΔF expressing H. armigera FAR), produced 4.30±2.33 mg/L Z9-16OH and 450±80 mg/L Z9-16Acid using a semi-defined medium (C:N ratio of ˜80).
Results
Strain Construction
Evidence in the literature suggests both insect desaturases and FARs are localized in the membrane of the endoplasmic reticulum with active sites oriented towards the cytoplasm. Of the functional variants, the Z11 desaturase from H. zea and the FAR from H. armigera (FAR amino acid sequence set forth in SEQ ID NO: 41) were selected, one hypothesis being that using enzymes from the same genus (Helicoverpa) could better conserve protein-protein interactions that may occur in the ER membrane.
Two new constructs were ordered from Genscript and cloned into the previously assembled H. zea desaturase plasmid, pPV0199. Two FAR synthons with either the EXP1 or TAL1 promoter from Y. lipolytica were cloned into this expression cassette.
One dual expression plasmid (with EXP1 promoter) was transformed into the parent strain SPV300 (H222 Δpox1 Δpox2 Δpox3 Δpox4 Δpox5 Δpox6 Δadh1 Δadh2 Δadh3 Δadh4 Δadh5 Δadh6 Δadh7 Δfao1 Δura3). Two different competent cell preparations of the same parent strain were transformed to study variability in strain performance resulting from competent cell preparation. Approximately 25% of URA+clones were confirmed to be targeted integrants at the XPR2 locus (20% for preparation 1, 33% for preparation 2). Two clones from Comp. Cell Preparation 1 and all eight targeted clones from Comp. Cell Preparation 2 were selected for screening in the functional expression assay.
The second dual expression plasmid (with TAL1 promoter) was integrated into the same parent strain (SPV300). Twenty-three colonies were screened by check PCR and 11 were found to be targeted integrants (48%). Nine integrants were selected for screening in the functional expression assay.
The construct of SPV471 (H222 ΔPΔAΔF expressing H. armigera FAR) was described previously.
Z11-16OH Functional Expression Assay
An in vivo, 24-well plate assay was used to evaluate production of Z11-16OH. The assay was based on designs used for screening desaturase and reductase variants as well as conditions used to increase fatty acid accumulation. A rich medium (YPD) and a semi-defined medium were used with 10 g/L methyl palmitate supplemented as bioconversion substrate. The semi-defined medium had a C:N ratio of ˜80 and included 5 g/L glycerol and 60 g/L glucose (See Materials & Methods for further details).
The initial screen of strains harboring the H. zea desaturase driven by the TEF promoter and the H. armigera FAR (FAR amino acid sequence set forth in SEQ ID NO: 41) driven by the EXP1 promoter confirmed that the presence of FAR was required to produce Z11-16OH. No hexadecenol was observed from both the parent and desaturase-only control strains under any condition. Under both media conditions Z11-16OH and to a lesser extent Z9-16OH were detected from clones expressing the full desaturase-reductase pathway. When the conversion was completed in rich medium, 0.26±0.09 mg/L Z11-16OH and 0.06±0.01 mg/L Z9-16OH were produced (
The lipid profiles of the full pathway clones were also quantified. For simplicity the 16 carbon fatty acid species are plotted for select clones in
Strains using the second dual expression cassette (pTAL-Ha_FAR) were assayed under the same Semi-Defined medium condition used to evaluate the pEXP clones. Nine pTAL clones were assayed against SPV300 (parent), SPV575 (pEXP-Ha_FAR Clone 4), and SPV578 (pEXP-Ha_FAR Clone 17) controls. As expected, no alcohol products were observed from the negative control. Alcohol titers from pEXP positive control strains replicated results observed in the initial assay of pEXP clones (
The lipid profiles of all strains in the second (pTAL) full pathway screen were also quantified. For simplicity the 16 carbon fatty acid species are plotted in
Z9-16OH Functional Expression Assay
An in vivo, flask scale assay was used to test for Z9-16OH production. The parent control strain, H222 ΔPΔAΔF (SPV300), was compared to a strain expressing H. armigera FAR which relied on native Z9 desaturase activity to synthesize the Z9-16CoA precursor (SPV471). Biomass was generated through a YPD seed culture, mimicking the plate assay. Bioconversion flasks were inoculated at an initial OD600=1 or OD600=4 into the same Semi-Defined C:N=80 medium used in the Z11-16OH plate assay (See Materials & Methods for details). As expected, control flasks did not produce detectable Z9-16OH while SPV471 flasks produced up to 4.30±2.23 mg/L after 24 hours of incubation (
Conclusions
Combining expression of Helicoverpa Z11 desaturase and fatty acyl-CoA reductase led to production of Z11-16OH in Y. lipolytica H222 ΔPΔAΔF (SPV300) at titers >1 mg/L.
High C:N ratio conditions improved Z11-16OH titer relative to a rich medium condition.
Under lipid accumulating conditions the combination of native Z9 desaturase and H. armigera FAR activities are sufficient for synthesis of >1 mg/L Z9-16OH.
Titers are increased, for example, by deleting pathways consuming fatty alcohol products and/or fatty acid precursors; identifying FAR variants which exhibit higher turnover rate than H. armigera FAR; and/or increasing pathway copy number.
Key undesired byproducts are identified.
The possibility that some of the fatty alcohol product is converted into fatty acetate by the activity of one or more endogenous acetyltransferases is explored.
Improved host strains are engineered to eliminate the w-oxidation pathway and components of the lipid storage pathway.
Materials & Methods
Strain Construction
All desaturase and reductase genes were ordered from Genscript. Homo sapiens codon optimization was used (Genscript algorithm). The newly synthesized expression cassette was subcloned into pPV199 by Genscript using the SapI restriction site. Plasmids were transformed and prepped from E. coli EPI400 using the Zyppy Plamsid Miniprep Kit (Zymo Research, Irvine, CA). Plasmids were digested with PmeI (New England Biolabs, Ipswich, MA) and purified by gel extraction using Zymoclean Gel DNA recovery Kit (Zymo Research, Irvine, CA). DNA was further concentrated using Clean and Concentrator Kit (Zymo Research, Irvine, CA). Approximately ˜1-2 μg of DNA was transformed using Frozen-EZ Yeast Transformation II Kit (Zymo Research, Irvine, CA). The manufacturer's protocol was modified as follows: A 2 ml YPD seed culture was inoculated at 9 am the day before competent cell preparation. The seed was grown 8 hours (until 5 pm) before 40 ml of YPD in a 250 ml baffled shake flask (or 20 ml in a 125 ml baffled flask) was inoculated to an initial OD600 of 0.0005. The culture was incubated at 28° C. and 250 rpm ˜24 hours. Cells were harvested at an OD600=0.5-1. Instead of resuspending 10 ml of culture in 1 ml of Solution 2 as in the manufacturer's instructions (OD600˜10), 10 ml of SPV140 culture was resuspended in 0.5 ml (OD600˜20-30). All Solution 2 aliquots were slowly frozen to −80° C. by placing the tubes in a closed Styrofoam box before putting in the −80° C. freezer. 50 μl aliquots of competent cells in 1.7 ml Eppendorf tubes were thawed on ice, DNA eluted in water was added directly to the cells, and 500 μl of Solution 3 was used to suspend the cells with gentle pipetting. Tubes were incubated at 28° C. for 3 hours with gentle vortexing every 30 minutes. The entire transformation mixture was plated on CM glucose-ura agar plates. Positive integrants were found to be site-specific and genotyping was conducted by check PCR.
Z11-16OH Functional Expression Assay
Positive isolates were repatched onto YPD, grown overnight, and then stored at 4° C. Strains were inoculated from patch plates into 2 ml of YPD in 24 deepwell plates (square well, pyramid bottom). Replicate inoculations were made from each patch. Negative control strains were struck out on YPD from glycerol stocks and individual colonies were used to inoculate. Deepwell plates were incubated at 28° C. and 250 rpm in the Infors Multitron refrigerated flask shaker for 24 hrs. After 24 hrs of incubation, a 0.85 ml volume of each culture was pelleted by centrifugation at 800×g. Each pellet was resuspended in either 2 ml of YPD or Semi-defined medium (described in Table 17 below). 10 g/L methyl palmitate (pre-warmed to ˜50° C.) was added to cultures. All cultures were incubated for 48 hours before endpoint sampling. Final cell densities were measured with the Tecan Infinite 200pro plate reader. 1.5 ml (alcohol analysis) or 500 μl (lipid analysis) was transferred to 1.7 ml microcentrifuge tubes and pelleted. Supernatant was transferred to clean tubes and samples were processed as described below.
Z9-16OH Functional Expression Assay
SPV300 (negative control) and SPV471 were struck out onto YPD agar plates, grown overnight, and then stored at 4° C. Strains were inoculated from colonies into 2 ml of YPD and incubated at 28° C. and 250 rpm in 14 ml round bottom culture tubes for ˜8 hours. After incubation, 2 ml of culture was used to inoculate 20 ml of YPD in a 125 ml baffled shake flask. Shake flasks were incubated 24 hrs at 28° C. and 250 rpm. After incubation, cell density in shake flasks was measured using a Tecan Infinite 200pro plate reader. An appropriate volume of culture was pelleted in order to resuspend cells in 25 ml of Semi-defined C:N=80 medium (see Table 17 above) at an initial OD600=1 (˜1 gDCW/L) or 4 (˜4gDCW/L). The resuspended culture was added to 250 ml baffled shake flasks. Neat methyl palmitate was added at 10 g/L final concentration after pre-heating to 50° C. After substrate addition, flasks were incubated at 28° C. and 250 rpm for two days. At 12, 18, 24, 36, 42, and 48 hours 500 μl (lipid analysis) and 1.5 ml (alcohol analysis) samples were taken in 1.7 ml microcentrifuge tubes. Samples were pelleted and the supernatant was transferred to a clean microcentrifuge tube.
Metabolite Extraction and GC-MS Detection
Alcohol Analysis
The pelleted cells (in 1.5 mL plastic tubes), usually about 10 mg to 80 mg, were resuspended in methanol containing 5% (w/w) of sodium hydroxide. The alkaline cell suspension was transferred into a 1.8 mL crimp vial. The mixture was heated for 1 h in the heat block at 90° C. Prior to acidification with 400 μL 2.5 N HCl the vial was allowed to cool to room temperature. 500 μL chloroform containing 1 mM methyl heptadecanoate were added and the mixture was shaken vigorously, then both aqueous and organic phase were transferred into a 1.5 mL plastic tube. The mixture was centrifuged at 13,000 rpm, afterwards 450 μL of the organic phase were transferred into a GC vial. The organic phase was evaporated in a heat block at 90° C. for 30 min. The residue was dissolved in 50 μL N,O-Bis(trimethylsilyl)trifluoroacetamide containing 1% trimethylchlorosilane. Prior to transfer into glass inserts the mixture was heated 5 min at 90° C. The samples were analyzed by GC-MS (Table 18).
Lipid Analysis
Total lipid composition was based on modified procedures by Moss et al. (1982) and Yousuf et al (2010). The pelleted cells (in 1.5 mL plastic tubes), usually about 10 mg to 80 mg, were resuspended in methanol containing 5% (w/w) of sodium hydroxide. The alkaline cell suspension was transferred into a 1.8 mL glass crimp GC-vial. The mixture was heated for 1 h in the heat block at 90° C. Prior to acidification with 400 μL 2.5 N HCl, the vial was allowed to cool to room temperature. 500 μL chloroform containing 1 mM methyl heptadecanoate were added and the mixture was shaken vigorously, then both aqueous and organic phase were transferred into a 1.5 mL plastic tube. The mixture was centrifuged at 13,000 rpm, afterwards 450 μL of the organic phase was transferred into a new 1.8 mL glass screw-cap GC-vial. After cooling to room temperature residual fatty acid methyl esters and free fatty acids were dissolved and derivatized in methanol containing 0.2 M TMSH (trimethylsulfonium hydroxide)(Table 19).
Background and Rationale
Yarrowia lipolytica was engineered to produce Z11-14Acid, the precursor to target Lepidoptera pheromone Z11-14Ac.
A library of 73 desaturases was chosen to target potential pheromones including Z11-14Ac, Z7-12Ac, Z9E12-14Ac, E8E10-C12OH and Z9E11-14Ac. All desaturases were tested in the H222 ΔPΔAΔF (SPV300) background.
Eleven desaturases were identified from literature to have Δ11 activity (DST001-DST009, DST030, and DST039, Table 20). All desaturases were screened by feeding either methyl palmitate (C16), methyl myristate (C14), or methyl laurate (C12) as substrate, and full product profiles were determined by GC analysis.
The resulting activity of the purported Δ11 desaturase library, and other desaturases shown to produce Δ11 compounds, specifically Z11-14Acid, is discussed.
Results
Up to 69 mg/L Z11-14Acid production was observed when feeding 2 g/L methyl myristate to the desaturase library (
Proof-of-concept of Z11-14Acid synthesis is shown. Attempts were made to identify enzymes that had improved Z11-16Acid titer or product specificity over Helicoverpa zea DST (1.05 g/L Z11-16Acid; 69 mg/L Z11-14Acid). While there were no desaturases that had higher production than Hz DST (SPV459), DST003 (SEQ ID NO: 39) had similar production phenotypes to the HzDesat strain, and DST002 and DST005 had similar Z11-16Acid production with reduced Z11-14Acid. The desaturase in DST006 is genetically equivalent to the H. zea desaturase expressed in SPV459 and served as a library control. DST006 produced equivalent levels of Z11-16Acid when fed methyl palmitate; however this strain produced a lower titer of Z11-14Acid on methyl myristate. Genetic variation in strain background may account for the observed difference.
DST039 (A. transitella) was previously screened under different conditions. In rich media, Z11-16Acid production with the native A. transitella coding sequence was not observed. The H. sapiens optimized sequence was tested and still no activity was observed with the rich medium condition. In this screen, DST039 was tested in nitrogen limited condition with Hs optimized sequence and resulted in 235 mg/L production of Z11-16Acid and 21 mg/L Z11-14Acid on the relevant substrates.
All products from DST008 (Drosophila melanogaster) or DST009 (Spodoptera littoralis) in the SPV300 background were not observed.
Summary
Z11-14Acid production was observed in ten desaturases with titers ranging from 16 mg/L to 69 mg/L (2 g/L methyl myristate fed).
H. zea DST (SEQ ID NO: 54) remained the best Z11-16Acid producer (>1 g/L when fed with methyl palmitate).
DST003 (S. inferens, SEQ ID NO: 39) has the most similar phenotype to H. zea DST.
DST002 (S. litura) and DST005 (O. nubilalis) are more specific than H. zea DST for Z11-16Acid production (reduced Z11-14Acid production).
DST001 (A. velutinana), DST004 (M. sexta), and DST039 (A. transitella) are more specific than H. zea DST for Z11-14Acid production.
Conclusions
Z11-14Acid can be produced with the heterologous expression of specific desaturases in Yarrowia lipolytica when feeding methyl myristate.
Multiple copies of desaturase (identical or combination of sequences) are integrated in improved strain backgrounds for increased Z11-14Acid titer, product specificity, and genetic stability.
Materials & Methods
Library Generation
Desaturase sequences were provided to Genscript for codon optimization (Homo sapiens expression organism) cloning into pPV266 (XPR2 locus integration vector with TEF promoter and terminator) using PacI/SapI restriction digestion. Lyophilized DNA was provided as well as EPI400 agar stabs. Desaturase constructs are listed in Table 21.
Constructs were linearized using PmeI restriction enzyme and directly transformed into host strain SPV300. Transformants were verified by check PCR using primers outside of the XPR2 integration junction and within the pTEF promoter.
Plasmid Digest
˜10 μg of lyophilized DNA was ordered from Genscript. DNA was resuspended in 50 μL water for a final concentration of ˜200 ng/μL. 10 μL of DNA was mixed with 1.25 μL 10× CutSmart Buffer and 1.25 μL PmeI restriction enzyme (12.5 μL reaction volume). The reaction was incubated in the PCR machine for 1.5 hours at 37° C. and heat inactivated at 65° C. for 30 minutes.
Transformation
SPV300 competent cells were grown by inoculating a YPD culture at 0.001 OD in a baffled flask and growing until 0.5-1.0 OD. Cells were harvested at 800×g and washed with 0.25× volume of Solution 1 from the Zymo Frozen-EZ Transformation II Kit for Yeast. Cells were resuspended in Solution 2 at 1000× concentration of the original culture volume and slowly frozen at −80° C. while insulated in a styrofoam container (frozen cells may have better transformation efficiency over fresh). 50 μL of cells were first mixed with the 12.5 μL digestion reaction (no cleanup necessary), and then with 500 μL Solution 3. Transformations were incubated for 3 hours at 28° C. without shaking, after which the full transformation mixture was plated to appropriate selective agar media. Petri dishes were incubated for 3-4 days before the appearance of colonies.
Check PCR
Transformation colonies were picked to 7 μL water in a PCR plate. 5 μL of cells were patched by multichannel to selective omni trays and grown overnight. The remaining 2 μL of cells were microwaved for 2 minutes before adding 15 μL of PCR master mix.
PCR Cycle:
Colony Patching
Positive clones were re-patched to YPD omni trays in 24-well format including assay controls. Omni trays were grown overnight at 28° C. and used to inoculate bioassay cultures.
Bioassay
Positive transformants (N=4 clones per construct) were inoculated into 1 mL YPD in a 24-well culture plate and incubated for 24 hours in the Infors HT Mulitron Pro at 28° C. with 1000 rpm shaking. Cells were pelleted at 800×g and resuspended in S2 media with 5 μL substrate (˜2 g/L concentration). 250 μL of culture was sampled into glass crimp top vials after 48 hours of bioconversion.
S2 Media
2 g/L Yeast Extract, 1 g/L Peptone, 0.1M Phosphate buffer, 1.7 g/L YNB w/o aa,NH4, 60 g/L Glucose, 5 g/L Glycerol
GC Sample Processing
Front Inlet/Detector:
Back Inlet/Detector:
TMSH: Trimethylsulfonium Hydroxide (0.2 mol/L in Methanol)—VWR TCT1576-025ML
Example 11 Production of Z11-16OH in Yarrowia lipolytica Using Increased Copy Number of or Engineered Variant Fatty Alcohol Forming Fatty Acyl-CoA Reductases (FARs)Background and Rationale
Engineering the microbial production of unsaturated insect fatty alcohols requires the functional expression of a synthetic pathway. One such pathway comprises a transmembrane desaturase to mediate the conversion of fatty acyl-CoA into regio- and stereospecific unsaturated fatty acyl-CoA. An alcohol-forming reductase (FAR) complements the synthetic pathway to produce the respective fatty alcohol.
In some insects species the respective FAR enzymes are activated via site specific dephosphorylation (Jurenka, R. & Rafaeli, A. Regulatory Role of PBAN in Sex Pheromone Biosynthesis of Heliothine Moths. Front. Endocrinol. (Lausanne). 2, 46 (2011); Gilbert, L. I. Insect Endocrinology. (Academic Press)). Phosphorylation of heterologously expressed FAR enzymes in Y. lipolytica may lead to inactivation, and result in low fatty alcohol titers. A bioinformatic approach was used to predict phosphorylated residues within HaFAR.
Alanine substitution of serine and threonine residues has been shown to abolish phosphorylation (Shi, S., Chen, Y., Siewers, V. & Nielsen, J. Improving Production of Malonyl Coenzyme A-Derived Metabolites by Abolishing Snf1-Dependent Regulation of Accl. mBio 5, (2014)). Thus, in addition to increasing FAR gene copy number, the impact of alanine substitutions of several serine residues of an HaFAR enzyme (HaFAR amino acid sequence set forth in SEQ ID NO: 41 and HaFAR nucleotide sequence set forth in SEQ ID NO: 90) on Z11-16OH titer was tested.
Approach
A second copy of human codon-optimized H. armigera FAR gene (HaFAR) was introduced into the chromosome of the Z11-16OH producer parent strain. In parallel, the effects of introducing a copy of a mutated H. armigera FAR gene variant towards Z11-16OH production improvement were also explored. Seven mutated variants were designed with the aim to increase FAR activity by potentially relieving the requirement for dephosphorylation (as observed in some insect species). To this end, several potential phosphorylation sites within the amino acid sequence of the H. armigera FAR were identified, and replaced with alanine.
Results
Determination of Phosphorylated Sites in HaFAR
The server at world wide web address: cbs.dtu.dk/services/NetPhos/( ) predicts potential phosphorylation sites based on a database (world wide web address: phospho.elm.eu.org/about.htmL) of experimentally verified phosphoproteins based on 17 kinases: ATM, CKI, CKII, CaMII, DNAPK, EGFR, GSK3, INSR, PKA, PKB, PKC, RSK, SRC, cdc2, cdk5 and p38MAPK.
The software program predicted 22 serine, 11 threonine and 10 tyrosine as potential phosphorylation sites in H. armigera FAR (
Next, a tailored prediction program to determine phosphorylation sites of the H. armigera FAR upon expression in yeasts was applied (world wide web address: cbs.dtu.dk/services/NetPhosYeast/; Blom, N., Gammeltoft, S. & Brunak, S. Sequence and structure-based prediction of eukaryotic protein phosphorylation sites1. J. Mol. Biol. 294, 1351-1362 (1999)). Since in yeast no tyrosine kinases have been identified yet (Ingrell, C. R., Miller, M. L., Jensen, O. N. & Blom, N. NetPhosYeast: prediction of protein phosphorylation sites in yeast. Bioinforma. 23, 895-897 (2007)), the yeast-specific software only considers serine and threonine as potential phosphorylation sites. It was striking that both programs predicted the same 11 serine residues to be phosphorylated. In contrast, the yeast specific analysis tool did not predict any phosphorylated threonine residues (
In a first experiment a small library consisting of 7 Ser to Ala point mutants was tested (Table 22). Three predicted phosphorylation sites were not considered for mutagenesis (position Ser301, Ser386, Ser416 scored barely above the threshold). For the remaining 7 serine residues, alanine substitutions were introduced.
The HaFAR library was custom synthesized, and subcloned into plasmid pPV234 for expression under TEF promoter, and XPR2 terminator at the AXP locus. Linearized constructs were transformed into strain Y. lipolytica H222ΔPΔAΔF SPV603 which expresses H. zea Z11 desaturase (SEQ ID NO: 54) combined with the H. armigera FAR (SEQ ID NO: 41). This approach determined the impact of a second copy e.g. protein expression in addition to the impact of the individual point mutations.
Four individual clones of positive integrants were tested. Cultivation was performed in 24 well plates. Briefly, 2 mL YPD was inoculated from patches of individual clones and incubated for 24 h at 28° C., 1000 rpm. After 24 h OD600 was measured and the cells were centrifuged at 1000 rpm (Table 23). The cell pellets were resuspended in 1 mL S2 media and 10 g/L methyl palmitate was added. The cells were incubated for 18 h at 28° C., 1000 rpm. Cultivations were stored at −20° C. until analyzed. Extraction and analysis was performed according to previously established standard protocols using GC-FID.
As shown in
Overall, these results suggest that residue 195 and 418 are important for increasing HaFAR activity in Y. lipolytica. Their substitutions to alanine may inhibit phosphorylation; therefore, a dephosphorylation mechanism was not required for their enhanced activity. Among other explanations, the activating effect of the serine to alanine mutation could be from improved protein folding, increased stability or higher protein expression.
In addition, several fatty acid species were quantified to determine whether the increased Z11-16OH production led to Z11-16Acid depletion. The standard deviations for some samples were very high (
Chromosomal integration of an additional HaFAR into the parent strain increased the Z11-16OH titer from ˜20 mg/L to 40 mg/L in shake flask experiments.
When HaFAR (Ser195A1a) or HaFAR (Ser418A1a) was introduced, Z11-16OH titer increased from ˜40 mg/L to ˜120 mg/L or ˜80 mg/L, respectively in shake flask experiments.
The pathway intermediate Z11-16Acid for mutant strains SPV910 harboring HaFAR (Ser195A1a), and SPV914 harboring HaFAR (Ser418Ala) accumulated at ˜100 mg/L less than the parent strain SPV603.
Conclusions
The addition of another H. armigera FAR gene into Z11-16OH producer strain marginally increased titer. Introduction of mutated FARs, however, significantly improved Z11-16OH by up to ˜7×. This suggests that Y. lipolytica phosphorylates FAR enzymes, and that FAR dephosphorylation is a bottleneck for its full activity. The designed mutations in HaFAR may relieve its requirement for dephosphorylation to convert Z11-16Acid into Z11-16OH. It is also possible that the designed mutations improved FAR activity through a dephosphorylation-independent mechanism. Shake flask experiments suggest a direct correlation between biomass, time and fatty alcohol titers.
Second generation strains are created for further improvement in Z11-16OH by, for example, eliminating lipid storage pathways (e.g. diacylglycerol acetyltransferase (DGAT) gene deletions), and/or eliminating byproduct (hydroxyacid, diacid) pathways.
Materials & Methods
Marker Rescue of SPV603
30 mL of CM minus uracil were inoculated with SPV603 in a 250 mL baffled shake flask. The culture was incubated in a bench top shaker at 250-300 rpm and 28° C. The following morning the OD600 of the culture was measured. Cells were harvested at OD600: 0.6. Cells were pelleted in a falcon tube (800×g, 5 min). Supernatant was removed and wash in ½ volume (15 mL) of Solution 1 from the Zymo kit. Cells were pelleted again (800×g, 5 min) and resuspended in 200 μl of Solution 2. A 50 μl aliquot was used directly in a 1.7 mL Eppendorf tube. Remaining aliquots were stored at −80° C. 10 μl of DNA (1-2 μg) was added and gently mixed with cells. 500 μl of Solution 3 was added and mixed gently. Transformation mix was incubated for ≥3 hours in 28° C. plate incubator subsequently washed with 2 mL deionized water (autoclaved). Recovery was performed in 2 mL YPD o/n at 28° C. The transformation mix was plated directly on selection plate. Clones were picked and repatched on URA minus FOA 0.1% and YPD plates. URA removal was confirmed using standard PCR protocols.
Construction of HaFAR Integration Cassette
The HaFAR library was custom synthesized with human codons (Genscript), and subcloned into plasmid pPV234 using the restriction site SpeI and NotI for expression under TEF promoter, and XPR2 terminator at the AXP locus. Linearized constructs were transformed into strain Y. lipolytica H222ΔPΔAΔF SPV603 which expresses H. zea Z11 desaturase (SEQ ID NO: 54) combined with the H. armigera FAR (SEQ ID NO: 41). Amino acid sequences for the variant FARs are set forth in SEQ ID NOs: 42-48. Nucleotide sequences for the variant FARs are set forth in SEQ ID NOs: 83-89.
Cultivation of HaFAR Mutant Library in 24 Well Plates
In a first round four individual isolate from each transformation was tested. Cultivation was performed in 24 well plates. 2 mL YPD was inoculated from patches of individual clones and incubated for 24 h at 28° C., 1000 rpm. After 24 h OD600 was measured and the cells were centrifuged at 1000 rpm. The cell pellets were resuspended in 1 mL S2 media and 10 g/L methyl palmitate was added. The cells were incubated for 24 h at 28° C., 1000 rpm. Cultivations were stored at −20° C. until analyzed. Next, four clones of the best performing positive integrant were tested. Cultivation was performed in 24 well plates. 2 mL YPD was inoculated from patches of individual clones and incubated for ˜28 h at 28° C., 1000 rpm. After 28 h OD600 was measured and the cells were centrifuged at 1000 rpm. The cell pellets were resuspended in 1 mL S2 media and 10 g/L methyl palmitate was added. The cells were incubated for 18 h at 28° C., 1000 rpm. Cultivations were stored at −20° C. until analyzed.
Cultivation of HaFAR Mutant Library in Shake Flasks
Shake Flask Experiment 1: Time-Course Analysis of Fatty Alcohol and Fatty Acid Titers During Shake Flask Cultivation
In a first step a shake flask experiment of the respective strains was performed to analyze fatty alcohol and fatty acid formation over time. The best performing clone of the strains expressing HaFAR, HaFAR S195A and HaFAR S418A were tested. 10 mL YPD was inoculated from patches of individual clones and incubated for 24 h at 28° C., 250 rpm in a 125 mL shake flask. Next, 25 mL YPD were inoculated with 5 mL starter culture in 500 mL shake flasks and incubated for 24 h at 28° C., 250 rpm. The cells were harvested and the pellets were resuspended in 15 mL S2 media and 10 g/L methyl palmitate was added. OD600 was measured and subsequently methyl palmitate was added. The cells were incubated for 72 h at 28° C., 1000 rpm. 4×0.5 mL samples were taken every 24 h and stored at −20° C. until analyzed according to a previously established standard protocol (
The OD600 for the strains were: SPV910-10.2, SPV914-11.5 and SPV916-12.3.
Shake Flask Experiment 2: Impact of Increased Biomass on Bioconversion
The time-course experiment revealed a decrease in fatty alcohols over time. Thus, sampling was reduced to 20 h. In previous 24 well plate bioconversion experiments the measured biomass was ˜2× higher compared to the shake flask experiment. Next a shake flask experiment of the respective strains was performed to analyze the impact of biomass on fatty alcohol titers. The overall incubation time was reduced and the biomass was increased during bioconversion phase. The best performing clone of the strains expressing HaFAR S195A and HaFAR S418A were tested. 20 mL YPD was inoculated from patches of individual clones and incubated for 28 h at 28° C., 250 rpm in a 500 mL shake flask. The cells were harvested and the pellets were resuspended in 10 mL S2 media and 10 g/L, OD600 was measured and subsequently methyl palmitate was added. The cells were incubated for 20 h at 28° C., 1000 rpm. 4×0.5 mL samples were taken and analyzed according to a previously established standard protocol (
The OD600 for the strains were: SPV910-24.9 and SPV914-24.7.
Metabolite Extraction
Cell pellets were resuspended with 500 μL 5% NaOH (in methanol) then heated for 1 hour at 85° C. Samples were cooled down and then acidified by adding 400 μL 5N HCl. Next, 500 μL chloroform (containing 1 mM C17:0 heptadecanoate internal standard) were added to samples. Samples were mixed vigorously then spun down for 2 minutes at 13,000 RPM using a table top centrifuge. 450 μL chloroform were transferred to a new vial then evaporated at 85° C. for ˜15 minutes. Samples were then resuspended in 50 μL BSTFA (and were now ready for GC analysis). After analysis of the fatty alcohols samples were diluted ˜1:10 and rerun on the GC to improve peak separation of the fatty acid peaks.
Quantification
All quantifications were based on the concentration of the internal standard. The concentration of the internal standard C17ME is 1 mM. The final concentration of fatty alcohols as well as fatty acids were calculated based on the effective carbon number compared to the internal standard.
Example 12 Production of Fatty Alcohol and Fatty Acid in Yarrowia lipolytica Using Second Generation Strains Engineered to Eliminate Lipid Storage Pathways or Using Additional Variant Fatty Alcohol Forming Fatty Acyl-CoA Reductases (FARs)The impact of an endogenous diacyl glycerol acyltransferase deletion selected from the group consisting of YALI0E32791g (DGA1) and YALI0D07986g (DGA2) was tested. Each dga gene was deleted individually in the strain SPV735 (ΔURA, ΔLeu, leu2::pTEF-HZ_Z11_desat_Hs-tXPR2_loxP). Subsequently, the individual selection marker in each deletion strain SPV957 (Δdga1, ΔLeu, leu2::pTEF-HZ_Z11_desat_Hs-tXPR2_loxP) and SPV959 (Δdga2 ΔURA, leu2::pTEF-HZ_Z11_desat_Hs-tXPR2_loxP) according to standard procedures was performed. The resulting strain SPV1053 (Δdga1 ΔURA, ΔLeu, leu2::pTEF-HZ_Z11_desat_Hs-tXPR2_loxP) and SPV1054 (Δdga2 ΔURA, ΔLeu, leu2::pTEF-HZ_Z11_desat_Hs-tXPR2_loxP) were used to transform HaFAR and HaFARS195A. The formation of fatty alcohols compared to the strain SPV603 was tested in shake flasks (
Time-Course Analysis of Fatty Alcohol and Fatty Acid Titers During Shake Flask Cultivation
A copy of the enzyme HAFAR (SEQ ID NO: 41) or HaS195A (SEQ ID NO: 43) was introduced into the strain SPV1053 (Δdga1 ΔURA, ΔLeu, leu2::pTEF-HZ_Z11_desat_Hs-tXPR2_loxP) and SPV1054 (Δdga2 ΔURA, ΔLeu, leu2::pTEF-HZ_Z11_desat_Hs-tXPR2_loxP), each of which expresses H. zea Z11 desaturase (SEQ ID NO: 54) and is deleted for either DGA1 or DGA2 diacylglycerol acyltransferase.
Cultivation was performed as biological triplicates in shake flasks. The starting culture for each strain was grown in 50 mL YPD in 250 mL shake flask for 28 h at 250 rpm, 28° C. Cells were harvested via centrifugation at 800 g for 5 min at room temperature and resuspended in 30 mL S2 media. Cell suspensions were normalized to OD600: ˜8. Each starting culture was split in three individual 125 mL shake flasks. 10 g/L methyl palmitate was added. Cultures were incubated for 72 h. Sampling was performed every 24 h and OD600 for each shake flask was measured.
Extraction of cultivation samples. 0.5 mL of each sample were harvested directly into crimp vials. The crimp vials were centrifuged at 800 g for 5 min. The supernatant was removed and the samples were sealed and stored at −20° C. until analysis. Samples were resuspended in 500 μL methanol containing 5% KOH and incubated for 60 min at 65° C. to 85° C. 200 μL 5N HCL was added to each crimp vial after cool down step. 600 μL chloroform containing 1 mM C17Me were added and sealed again. Samples were mixed and centrifuged. 500 uL of the chloroform was transferred to a new GC vial and dried to completeness at 85° C. for 30 min. Samples were resuspend in 100 uL BSTFA and incubated at room temperature for 1 h. Samples were analyzed according to standard protocol using gas chromatography.
Fatty Alcohol Forming Fatty Acyl-CoA Reductase Library Screening of New Strains in 24 Well Plates
A single copy of each respective FAR enzyme from Table 24 was introduced into the strain SPV1054 (Δdga2 ΔURA, ΔLeu, leu2::pTEF-HZ_Z11_desat_Hs-tXPR2_loxP) expressing H. zea Z11 desaturase (SEQ ID NO: 54) and deleted for DGA2 diacylglycerol acyltransferase.
Cultivation was performed as biological quadruplicates in 24 well plates. 2 mL YPD were inoculated from a patch of each individual transformant. 24 well plates were incubated for 28 h at 250 rpm, 28° C. Cells were harvested via centrifugation at 800 g for 5 min at room temperature. Cells were resuspended in 1 mL S2+60 g/L glycerol and 10 g/L methyl palmitate was added. Cell were incubated for 96 h at 1000 rpm, 28° C.
Extraction of cultivation samples. 0.5 mL of each sample were harvested directly into crimp vials. The crimp vials were centrifuged at 800 g for 5 min. The supernatant was removed, and the samples were sealed and stored at −20° C. until analysis. Samples were resuspended in 500 μL methanol containing 5% KOH and incubated for 60 min at 65° C. to 85° C. 200 μL 5N HCL was added to each crimp vial after cool down step. 600 μL chloroform containing 1 mM C17Me were added and sealed again. Samples were mixed and centrifuged. 500 uL of the chloroform was transferred to a new GC vial and dried to completeness at 85° C. for 30 min. Samples were resuspend in 100 uL BSTFA and incubated at room temperature for 1 h. Samples were analyzed according to standard protocol using gas chromatography.
Additional FAR variants and additional bacterial FAR enzymes are listed in Table 25 and Table 26, respectively. These FAR enzymes are tested as described above for fatty alcohol and fatty acid production.
The foregoing detailed description has been given for clearness of understanding only and no unnecessary limitations should be understood there from as modifications will be obvious to those skilled in the art.
While the disclosure has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the disclosure following, in general, the principles of the disclosure and including such departures from the present disclosure as come within known or customary practice within the art to which the disclosure pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims.
INCORPORATION BY REFERENCEAll references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entireties for all purposes. The current application hereby incorporates by reference each of the following in its entirety: U.S. Provisional Application Ser. No. 62/257,054, filed Nov. 18, 2015, U.S. Provisional Application Ser. No. 62/351,605, filed Jun. 17, 2016, and PCT application no. PCT/US2016/062852, filed Nov. 18, 2016.
However, mention of any reference, article, publication, patent, patent publication, and patent application cited herein is not, and should not be taken as, an acknowledgment or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.
Further Embodiments of the InventionOther subject matter contemplated by the present disclosure is set out in the following numbered embodiments:
1. A recombinant Yarrowia lipolytica microorganism capable of producing a mono- or poly-unsaturated C6-C24 fatty alcohol from an endogenous or exogenous source of saturated C6-C24 fatty acid, wherein the recombinant Yarrowia lipolytica microorganism comprises:
-
- (a) at least one nucleic acid molecule encoding a fatty acyl desaturase having at least 95% sequence identity to a fatty acyl desaturase selected from the group consisting of SEQ ID NOs: 39, 54, 60, 62, 78, 79, 80, 95, 97, 99, 101, 103, and 105 that catalyzes the conversion of a saturated C6-C24 fatty acyl-CoA to a corresponding mono- or poly-unsaturated C6-C24 fatty acyl-CoA; and
- (b) at least one nucleic acid molecule encoding a fatty alcohol forming fatty acyl reductase having at least 95% sequence identity to a fatty alcohol forming fatty acyl reductase selected from the group consisting of SEQ ID NOs: 41-48, 57, 73, 75 and 77 that catalyzes the conversion of the mono- or poly-unsaturated C6-C24 fatty acyl-CoA from (a) into the corresponding mono- or poly-unsaturated C6-C24 fatty alcohol.
2. The recombinant Yarrowia lipolytica microorganism of embodiment 1, wherein the recombinant Yarrowia lipolytica microorganism comprises a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous enzymes that catalyzes a reaction in a pathway that competes with the biosynthesis pathway for the production of a mono- or poly-unsaturated C6-C24 fatty alcohol.
3. The recombinant Yarrowia lipolytica microorganism of embodiments 1 or 2, wherein the recombinant Yarrowia lipolytica microorganism comprises a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous enzyme selected from the following:
-
- (i) one or more acyl-CoA oxidase selected from the group consisting of YALI0E32835g (POX1), YALI0F10857g (POX2), YALI0D24750g (POX3), YALI0E27654g (POX4), YALI0C23859g (POX5), YALI0E06567g (POX6);
- (ii) one or more (fatty) alcohol dehydrogenase selected from the group consisting of YALI0F09603g (FADH), YALI0D25630g (ADH1), YALI0E17787g (ADH2), YALI0A16379g (ADH3), YALI0E15818g (ADH4), YALI0D02167g (ADH5), YALI0A15147g (ADH6), YALI0E07766g (ADH7);
- (iii) a (fatty) alcohol oxidase YALI0B14014g (FAO1);
- (iv) one or more cytochrome P450 enzyme selected from the group consisting of YALI0E25982g (ALK1), YALI0F01320g (ALK2), YALI0E23474g (ALK3), YALI0B13816g (ALK4), YALI0B13838g (ALK5), YALI0B01848g (ALK6), YALI0A15488g (ALK7), (YALI0C12122g (ALK8), YALI0B06248g (ALK9), YALI0B20702g (ALK10), YALI0C10054g (ALK11) and YALI0A20130g (Alk12); and
- (v) one or more diacylglycerol acyltransferase selected from the group consisting of YALI0E32791g (DGA1) and YALI0D07986g (DGA2).
4. The recombinant Yarrowia lipolytica microorganism of embodiments 1 or 2, wherein the recombinant Yarrowia lipolytica microorganism comprises a deletion of one or more endogenous enzyme selected from the following:
-
- (i) one or more acyl-CoA oxidase selected from the group consisting of YALI0E32835g (POX1), YALI0F10857g (POX2), YALI0D24750g (POX3), YALI0E27654g (POX4), YALI0C23859g (POX5), YALI0E06567g (POX6);
- (ii) one or more (fatty) alcohol dehydrogenase selected from the group consisting of YALI0F09603g (FADH), YALI0D25630g (ADH1), YALI0E17787g (ADH2), YALI0A16379g (ADH3), YALI0E15818g (ADH4), YALI0D02167g (ADH5), YALI0A15147g (ADH6), YALI0E07766g (ADH7);
- (iii) a (fatty) alcohol oxidase YALI0B14014g (FAO1);
- (iv) one or more cytochrome P450 enzyme selected from the group consisting of YALI0E25982g (ALK1), YALI0F01320g (ALK2), YALI0E23474g (ALK3), YALI0B13816g (ALK4), YALI0B13838g (ALK5), YALI0B01848g (ALK6), YALI0A15488g (ALK7), (YALI0C12122g (ALK8), YALI0B06248g (ALK9), YALI0B20702g (ALK10), YALI0C10054g (ALK11) and YALI0A20130g (Alk12); and
- (v) one or more diacylglycerol acyltransferase selected from the group consisting of YALI0E32791g (DGA1) and YALI0D07986g (DGA2).
5. The recombinant Yarrowia lipolytica microorganism of any one of embodiments 1-4, wherein the fatty acyl desaturase catalyzes the conversion of a saturated fatty acyl-CoA into a mono- or poly-unsaturated intermediate selected from Z9-14:Acyl-CoA, Z11-14:Acyl-CoA, E11-14:Acyl-CoA, Z9-16:Acyl-CoA, and Z11-16:Acyl-CoA.
6. The recombinant Yarrowia lipolytica microorganism of any one of embodiments 1-5, wherein the mono- or poly-unsaturated C6-C24 fatty alcohol is selected from the group consisting of Z9-14:OH, Z11-14:OH, E11-14:OH, Z9-16:OH, Z11-16:OH, Z11Z13-16:OH, and Z13-18:OH.
7. The recombinant Yarrowia lipolytica microorganism of any one of embodiments 1-6, wherein the recombinant Yarrowia lipolytica microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an alcohol oxidase or an alcohol dehydrogenase capable of catalyzing the conversion of the mono- or poly-unsaturated C6-C24 fatty alcohol into a corresponding C6-C24 fatty aldehyde.
8. The recombinant Yarrowia lipolytica microorganism of embodiment 7, wherein the alcohol dehydrogenase is selected from Table 3a.
9. The recombinant Yarrowia lipolytica microorganism of embodiments 7 or 8, wherein the C6-C24 fatty aldehyde is selected from the group consisting of Z9-14:Ald, Z11-14:Ald, E11-14:Ald, Z9-16:Ald, Z11-16:Ald, Z11Z13-16:Ald and Z13-18:Ald.
10. The recombinant Yarrowia lipolytica microorganism of any one of embodiments 1-9, wherein the recombinant Yarrowia lipolytica microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an acetyl transferase capable of catalyzing the conversion of the mono- or poly-unsaturated C6-C24 fatty alcohol into a corresponding C6-C24 fatty acetate.
11. The recombinant Yarrowia lipolytica microorganism of embodiment 10, wherein the acetyl transferase is selected from Table 5d.
12. The recombinant Yarrowia lipolytica microorganism of embodiments 10 or 11, wherein the C6-C24 fatty acetate is selected from the group consisting of Z9-14:Ac, Z11-14:Ac, E11-14:Ac, Z9-16:Ac, Z11-16:Ac, Z11Z13-16:Ac, and Z13-18:Ac.
13. The recombinant Yarrowia lipolytica microorganism of any one of embodiments 1-12, wherein the recombinant Yarrowia lipolytica microorganism further comprises:
-
- at least one endogenous or exogenous nucleic acid molecule encoding an alcohol oxidase or an alcohol dehydrogenase capable of catalyzing the conversion of the mono- or poly-unsaturated C6-C24 fatty alcohol into a corresponding C6-C24 fatty aldehyde; and
- at least one endogenous or exogenous nucleic acid molecule encoding an acetyl transferase capable of catalyzing the conversion of the mono- or poly-unsaturated C6-C24 fatty alcohol into a corresponding C6-C24 fatty acetate.
14. The recombinant Yarrowia lipolytica microorganism of embodiment 13, wherein the mono- or poly-unsaturated C6-C24 fatty aldehyde and C6-C24 fatty acetate is selected from the group consisting of Z9-14:Ac, Z11-14:Ac, E11-14:Ac, Z9-16:Ac, Z11-16:Ac, Z11Z13-16:Ac, Z13-18:Ac, Z9-14:Ald, Z11-14:Ald, E11-14:Ald, Z9-16:Ald, Z11-16:Ald, Z11Z13-16:Ald and Z13-18:Ald.
15. The recombinant Yarrowia lipolytica microorganism of any one of embodiments 1-14, wherein the fatty acyl desaturase does not comprise a fatty acyl desaturase comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 64, 65, 66 and 67.
16. The recombinant Yarrowia lipolytica microorganism of any one of embodiments 1-15, wherein the fatty acyl desaturase does not comprise a fatty acyl desaturase selected from an Amyelois transitella, Spodoptera littoralis, Agrotis segetum, or Trichoplusia ni derived desaturase.
17. A method of producing a mono- or poly-unsaturated C6-C24 fatty alcohol from an endogenous or exogenous source of saturated C6-C24 fatty acid, comprising: cultivating the recombinant Yarrowia lipolytica microorganism of any one of embodiments 1-16 in a culture medium containing a feedstock that provides a carbon source adequate for the production of the mono- or poly-unsaturated C6-C24 fatty alcohol.
18. The method of embodiment 17, wherein the mono- or poly-unsaturated C6-C24 fatty alcohol is selected from the group consisting of Z9-14:OH, Z11-14:OH, E11-14:OH, Z9-16:OH, Z11-16:OH, Z11Z13-16:OH, and Z13-18:OH.
19. The method of embodiments 17 or 18, wherein the recombinant Yarrowia lipolytica microorganism comprises a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous enzyme selected from the following:
-
- (i) one or more acyl-CoA oxidase selected from the group consisting of YALI0E32835g (POX1), YALI0F10857g (POX2), YALI0D24750g (POX3), YALI0E27654g (POX4), YALI0C23859g (POX5), YALI0E06567g (POX6);
- (ii) one or more (fatty) alcohol dehydrogenase selected from the group consisting of YALI0F09603g (FADH), YALI0D25630g (ADH1), YALI0E17787g (ADH2), YALI0A16379g (ADH3), YALI0E15818g (ADH4), YALI0D02167g (ADH5), YALI0A15147g (ADH6), YALI0E07766g (ADH7);
- (iii) a (fatty) alcohol oxidase YALI0B14014g (FAO1);
- (iv) one or more cytochrome P450 enzyme selected from the group consisting of YALI0E25982g (ALK1), YALI0F01320g (ALK2), YALI0E23474g (ALK3), YALI0B13816g (ALK4), YALI0B13838g (ALK5), YALI0B01848a (ALK6), YALI0A15488g (ALK7), (YALI0C12122g (ALK5), YALI0B06248g (ALK9), YALI0B20702g (ALK10), YALI0C10054g (ALK11) and YALI0A20130g (Alk12); and
- (v) one or more diacylglycerol acyltransferase selected from the group consisting of YALI0E32791g (DGA1) and YALI0D07986g (DGA2).
20. The method of embodiments 17 or 18, further comprising a step of recovering the mono- or poly-unsaturated C6-C24 fatty alcohol.
21. The method of embodiment 20, wherein said recovery step comprises distillation.
22. The method of embodiment 20, wherein said recovery step comprises membrane-based separation.
23. A method of producing a mono- or poly-unsaturated C6-C24 fatty aldehyde from an endogenous or exogenous source of saturated C6-C24 fatty acid, comprising: cultivating the recombinant Yarrowia lipolytica microorganism of any one of embodiment 1-16 in a culture medium containing a feedstock that provides a carbon source adequate for the production of the mono- or poly-unsaturated C6-C24 fatty aldehyde.
24. The method of embodiment 23, wherein the C6-C24 fatty aldehyde is selected from the group consisting of Z9-14:Ald, Z11-14:Ald, E11-14:Ald, Z9-16:Ald, Z11-16:Ald, Z11Z13-16:Ald and Z13-18:Ald.
25. The method of embodiments 23 or 24, further comprising a step of recovering the mono- or poly-unsaturated C6-C24 fatty aldehyde.
26. The method of embodiment 25, wherein said recovery step comprises distillation.
27. The method of embodiment 25, wherein said recovery step comprises membrane-based separation.
28. A method of producing a mono- or poly-unsaturated C6-C24 fatty acetate from an endogenous or exogenous source of saturated C6-C24 fatty acid, comprising: cultivating the recombinant Yarrowia lipolytica microorganism of any one of embodiments 1-16 in a culture medium containing a feedstock that provides a carbon source adequate for the production of the mono- or poly-unsaturated C6-C24 fatty acetate.
29. The method of embodiment 28, wherein the C6-C24 fatty acetate is selected from the group consisting of Z9-14:Ac, Z11-14:Ac, E11-14:Ac, Z9-16:Ac, Z11-16:Ac, Z11Z13-16:Ac, and Z13-18:Ac.
30. The method of embodiment 28, further comprising a step of recovering the mono- or poly-unsaturated C6-C24 fatty acetate.
31. The method of embodiment 28, wherein said recovery step comprises distillation.
32. The method of embodiment 28, wherein said recovery step comprises membrane-based separation.
33. A method of producing a mono- or poly-unsaturated C6-C24 fatty aldehyde and C6-C24 fatty acetate from an endogenous or exogenous source of saturated C6-C24 fatty acid, comprising: cultivating the recombinant Yarrowia lipolytica microorganism of any one of embodiments 1-16 in a culture medium containing a feedstock that provides a carbon source adequate for the production of the mono- or poly-unsaturated C6-C24 fatty aldehyde and C6-C24 fatty acetate.
34. The method of embodiment 33, wherein the mono- or poly-unsaturated C6-C24 fatty aldehyde and C6-C24 fatty acetate is selected from the group consisting of Z9-14:Ac, Z11-14:Ac, E11-14:Ac, Z9-16:Ac, Z11-16:Ac, Z11Z13-16:Ac, Z13-18:Ac, Z9-14:Ald, Z11-14:Ald, E11-14:Ald, Z9-16:Ald, Z11-16:Ald, Z11Z13-16:Ald and Z13-18:Ald.
35. A method of engineering a Yarrowia lipolytica microorganism that is capable of producing a mono- or poly-unsaturated C6-C24 fatty alcohol from an endogenous or exogenous source of saturated C6-C24 fatty acid, wherein the method comprises introducing into a Yarrowia lipolytica microorganism the following:
-
- (a) at least one nucleic acid molecule encoding a fatty acyl desaturase having at least 95% sequence identity to a fatty acyl desaturase selected from the group consisting of SEQ ID NOs: 39, 54, 60, 62, 78, 79, 80, 95, 97, 99, 101, 103, and 105 that catalyzes the conversion of a saturated C6-C24 fatty acyl-CoA to a corresponding mono- or poly-unsaturated C6-C24 fatty acyl-CoA; and
- (b) at least one nucleic acid molecule encoding a fatty alcohol forming fatty acyl reductase having at least 95% sequence identity to a fatty alcohol forming fatty acyl reductase selected from the group consisting of SEQ ID NOs: 41-48, 57, 73, 75 and 77 that catalyzes the conversion of the mono- or poly-unsaturated C6-C24 fatty acyl-CoA from (a) into the corresponding mono- or poly-unsaturated C6-C24 fatty alcohol.
36. The method of embodiment 35, wherein the method further comprises introducing into the Yarrowia lipolytica microorganism one or more modifications comprising a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous enzymes that catalyzes a reaction in a pathway that competes with the biosynthesis pathway for the production of a mono- or poly-unsaturated C6-C24 fatty alcohol.
37. The method of embodiment 35 or 36, wherein the Yarrowia lipolytica microorganism is MATA ura3-302::SUC2 Δpox1 Δpox2 Δpox3 Δpox4 Δpox5 Δpox6 Δfadh Δadh1 Δadh2 Δadh3 Δadh4 Δadh5 Δadh6 Δadh7 Δfao1::URA3.
38. The method of any one of embodiments 35-37, wherein the method further comprises introducing into the Yarrowia lipolytica microorganism one or more modifications comprising a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous enzyme selected from the following:
-
- (i) one or more acyl-CoA oxidase selected from the group consisting of YALI0E32835g (POX1), YALI0F10857g (POX2), YALI0D24750g (POX3), YALI0E27654g (POX4), YALI0C23859g (POX5), YALI0E06567g (POX6);
- (ii) one or more (fatty) alcohol dehydrogenase selected from the group consisting of YALI0F09603g (FADH), YALI0D25630g (ADH1), YALI0E17787g (ADH2), YALI0A16379g (ADH3), YALI0E15818g (ADH4), YALI0D02167g (ADH5), YALI0A15147g (ADH6), YALI0E07766g (ADH7);
- (iii) a (fatty) alcohol oxidase YALI0B14014g (FAO1);
- (iv) one or more cytochrome P450 enzyme selected from the group consisting of YALI0E25982g (ALK1), YALI0F01320g (ALK2), YALI0E23474g (ALK3), YALI0B13816g (ALK4), YALI0B13838g (ALK5), YALI0B01848g (ALK6), YALI0A15488g (ALK7), (YALI0C12122a (ALK5), YALI0B06248g (ALK9), YALI0B20702g (ALK10), YALI0C10054g (ALK11) and YALI0A20130g (Alk12); and
- (v) one or more diacylglycerol acyltransferase selected from the group consisting of YALI0E32791g (DGA1) and YALI0D07986g (DGA2).
39. The method of any one of embodiments 35-38, wherein the fatty acyl desaturase catalyzes the conversion of a fatty acyl-CoA into a mono- or poly-unsaturated intermediate selected from Z9-14:Acyl-CoA, Z11-14:Acyl-CoA, E11-14:Acyl-CoA, Z9-16:Acyl-CoA, and Z11-16:Acyl-CoA.
40. The method of any one of embodiments 35-39, wherein the mono- or poly-unsaturated C6-C24 fatty alcohol is selected from the group consisting of Z9-14:OH, Z11-14:OH, E11-14:OH, Z9-16:OH, Z11-16:OH, Z11Z13-16:OH, and Z13-18:OH.
41. The method of any one of embodiments 35-40, wherein the method further comprises introducing into or expressing in the recombinant Yarrowia lipolytica microorganism at least one endogenous or exogenous nucleic acid molecule encoding an alcohol oxidase or an alcohol dehydrogenase capable of catalyzing the conversion of the mono- or poly-unsaturated C6-C24 fatty alcohol into a corresponding C6-C24 fatty aldehyde.
42. The method of embodiment 41, wherein the alcohol dehydrogenase is selected from Table 3a.
43. The method of embodiment 41, wherein the C6-C24 fatty aldehyde is selected from the group consisting of Z9-14:Ald, Z11-14:Ald, E11-14:Ald, Z9-16:Ald, Z11-16:Ald, Z11Z13-16:Ald and Z13-18:Ald.
44. The method of any one of embodiment 35-43, wherein the method further comprises introducing into or expressing in the recombinant Yarrowia lipolytica microorganism at least one endogenous or exogenous nucleic acid molecule encoding an acetyl transferase capable of catalyzing the conversion of the mono- or poly-unsaturated C6-C24 fatty alcohol into a corresponding C6-C24 fatty acetate.
45. The method of embodiment 44, wherein the acetyl transferase is selected from Table 5d.
46. The method of embodiment 44, wherein the C6-C24 fatty acetate is selected from the group consisting of Z9-14:Ac, Z11-14:Ac, E11-14:Ac, Z9-16:Ac, Z11-16:Ac, Z11Z13-16:Ac, and Z13-18:Ac.
47. The method of any one of embodiments 35-46, wherein the method further comprises introducing into or expressing in the recombinant Yarrowia lipolytica microorganism:
-
- at least one endogenous or exogenous nucleic acid molecule encoding an alcohol oxidase or an alcohol dehydrogenase capable of catalyzing the conversion of the mono- or poly-unsaturated C6-C24 fatty alcohol into a corresponding C6-C24 fatty aldehyde; and
- at least one endogenous or exogenous nucleic acid molecule encoding an acetyl transferase capable of catalyzing the conversion of the mono- or poly-unsaturated C6-C24 fatty alcohol into a corresponding C6-C24 fatty acetate.
48. The method of any one of embodiments 35-47, wherein the mono- or poly-unsaturated C6-C24 fatty aldehyde and C6-C24 fatty acetate is selected from the group consisting of Z9-14:Ac, Z11-14:Ac, E11-14:Ac, Z9-16:Ac, Z11-16:Ac, Z11Z13-16:Ac, Z13-18:Ac, Z9-14:Ald, Z11-14:Ald, E11-14:Ald, Z9-16:Ald, Z11-16:Ald, Z11Z13-16:Ald and Z13-18:Ald.
49. The method of any one of embodiments 35-48, wherein the fatty acyl desaturase does not comprise a fatty acyl desaturase comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 64, 65, 66 and 67.
50. The method of any one of embodiments 35-49, wherein the fatty acyl desaturase does not comprise a fatty acyl desaturase selected from an Amyelois transitella, Spodoptera littoralis, Agrotis segetum, or Trichoplusia ni derived desaturase.
51. The method of any one of embodiments 17-22, wherein the mono- or poly-unsaturated C6-C24 fatty alcohol is converted into a corresponding C6-C24 fatty aldehyde using chemical methods.
53. The method of any one of embodiments 17-22, wherein the mono- or poly-unsaturated C6-C24 fatty alcohol is converted into a corresponding C6-C24 fatty acetate using chemical methods.
54. The method of embodiment 53, wherein the chemical methods utilize a chemical agent selected from the group consisting of acetyl chloride, acetic anhydride, butyryl chloride, butyric anhydride, propanoyl chloride and propionic anhydride in the presence of 4-N,N-dimethylaminopyridine (DMAP) or sodium acetate to esterify the mono- or poly-unsaturated C6-C24 fatty alcohol to the corresponding C6-C24 fatty acetate.
55. A recombinant microorganism capable of producing a mono- or poly-unsaturated ≤C18 fatty alcohol from an endogenous or exogenous source of saturated C6-C24 fatty acid, wherein the recombinant microorganism comprises:
-
- (a) at least one exogenous nucleic acid molecule encoding a fatty acyl desaturase that catalyzes the conversion of a saturated C6-C24 fatty acyl-CoA to a corresponding mono- or poly-unsaturated C6-C24 fatty acyl-CoA;
- (b) at least one exogenous nucleic acid molecule encoding an acyl-CoA oxidase that catalyzes the conversion of the mono- or poly-unsaturated C6-C24 fatty acyl-CoA from (a) into a mono- or poly-unsaturated ≤C18 fatty acyl-CoA after one or more successive cycle of acyl-CoA oxidase activity, with a given cycle producing a mono- or poly-unsaturated C4-C22 fatty acyl-CoA intermediate with a two carbon truncation relative to a starting mono- or poly-unsaturated C6-C24 fatty acyl-CoA substrate in that cycle; and
- (c) at least one exogenous nucleic acid molecule encoding a fatty alcohol forming fatty acyl reductase that catalyzes the conversion of the mono- or poly-unsaturated ≤C18 fatty acyl-CoA from (b) into the corresponding mono- or poly-unsaturated ≤C18 fatty alcohol.
56. The recombinant microorganism of embodiment 55, wherein the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an acyltransferase that preferably stores ≤C18 fatty acyl-CoA.
57. The recombinant microorganism of any one of embodiments 55-56, wherein the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an acyltransferase that preferably stores ≤C18 fatty acyl-CoA, and wherein the acyltransferase is selected from the group consisting of glycerol-3-phosphate acyl transferase (GPAT), lysophosphatidic acid acyltransferase (LPAAT), glycerolphospholipid acyltransferase (GPLAT) and diacylglycerol acyltransferases (DGAT).
58. The recombinant microorganism of any one of embodiments 55-57, wherein the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an acyltransferase that preferably stores ≤C18 fatty acyl-CoA, and wherein the acyltransferase is selected from Table 5b.
59. The recombinant microorganism of any one of embodiments 55-58, wherein the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an acylglycerol lipase that preferably hydrolyzes ester bonds of >C16, of >C14, of >C12 or of >C10 acylglycerol substrates.
60. The recombinant microorganism of any one of embodiments 55-59, wherein the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an acylglycerol lipase that preferably hydrolyzes ester bonds of >C16, of >C14, of >C12 or of >C10 acylglycerol substrates, and wherein the acylglycerol lipase is selected from Table 5c.
61. The recombinant microorganism of any one of embodiments 55-60, wherein the recombinant microorganism comprises a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous enzymes that catalyzes a reaction in a pathway that competes with the biosynthesis pathway for the production of a mono- or poly-unsaturated ≤C18 fatty alcohol.
62. The recombinant microorganism of any one of embodiments 55-61, wherein the recombinant microorganism comprises a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous enzyme selected from:
-
- (i) one or more acyl-CoA oxidase;
- (ii) one or more acyltransferase;
- (iii) one or more acylglycerol lipase and/or sterol ester esterase;
- (iv) one or more (fatty) alcohol dehydrogenase;
- (v) one or more (fatty) alcohol oxidase; and
- (vi) one or more cytochrome P450 monooxygenase.
63. The recombinant microorganism of any one of embodiments 55-62, wherein the recombinant microorganism comprises a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous acyl-CoA oxidase enzyme selected from the group consisting of Y. lipolytica POX1(YALI0E32835g), Y. lipolytica POX2(YALI0F10857g), Y. lipolytica POX3(YALI0D24750g), Y. lipolytica POX4(YALI0E27654g), Y. lipolytica POX5(YALI0C23859g), Y. lipolytica POX6(YALI0E06567g); S. cerevisiae POX1(YGL205W); Candida POX2 (CaO19.1655, CaO19.9224, CTRG_02374, M18259), Candida POX4 (CaO19.1652, CaO19.9221, CTRG_02377, M12160), and Candida POX5 (CaO19.5723, CaO19.13146, CTRG_02721, M12161).
64. The recombinant microorganism of any one of embodiments 55-63, wherein the recombinant microorganism comprises a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous acyltransferase enzyme selected from the group consisting of Y. lipolytica YALI0C00209g, Y. lipolytica YALI0E18964g, Y. lipolytica YALI0F19514g, Y. lipolytica YALI0C14014g, Y. lipolytica YALI0E16797g, Y. lipolytica YALI0E32769g, and Y. lipolytica YALI0D07986g, S. cerevisiae YBL011w, S. cerevisiae YDL052c, S. cerevisiae YOR175C, S. cerevisiae YPR139C, S. cerevisiae YNR008w, and S. cerevisiae YOR245c, and Candida 1503_02577, Candida CTRG_02630, Candida CaO19.250, Candida CaO19.7881, Candida CTRG_02437, Candida CaO19.1881, Candida CaO19.9437, Candida CTRG_01687, Candida CaO19.1043, Candida CaO19.8645, Candida CTRG_04750, Candida CaO19.13439, Candida CTRG_04390, Candida CaO19.6941, Candida CaO19.14203, and Candida CTRG_06209.
65. The recombinant microorganism of any one of embodiments 55-64, wherein the recombinant microorganism comprises a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous acylglycerol lipase and/or sterol ester esterase enzyme selected from the group consisting of Y. lipolytica YALI0E32035g, Y. lipolytica YALI0D17534g, Y. lipolytica YALI0F10010g, Y. lipolytica YALI0C14520g, and Y. lipolytica YALI0E00528g, S. cerevisiae YKL140w, S. cerevisiae YMR313c, S. cerevisiae YKR089c, S. cerevisiae YOR081c, S. cerevisiae YKL094W, S. cerevisiae YLL012W, and S. cerevisiae YLR020C, and Candida CaO19.2050, Candida CaO19.9598, Candida CTRG_01138, Candida W5Q_03398, Candida CTRG_00057, Candida CaO19.5426, Candida CaO19.12881, Candida CTRG_06185, Candida CaO19.4864, Candida CaO19.12328, Candida CTRG_03360, Candida CaO19.6501, Candida CaO19.13854, Candida CTRG_05049, Candida CaO19.1887, Candida CaO19.9443, Candida CTRG_01683, and Candida CTRG_04630.
66. The method of any one of embodiments 55-65, wherein the recombinant microorganism comprises a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous cytochrome P450 monooxygenases selected from the group consisting of Y. lipolytica YALI0E25982g (ALK1), Y. lipolytica YALI0F01320g (ALK2), Y. lipolytica YALI0E23474g (ALK3), Y. lipolytica YALI0B13816g (ALK4), Y. lipolytica YALI0B13838g (ALK5), Y. lipolytica YALI0B01848g (ALK6), Y. lipolytica YALI0A15488g (ALK7), Y. lipolytica YALI0C12122g (ALK8), Y. lipolytica YALI0B06248g (ALK9), Y. lipolytica YALI0B200702g (ALK10), Y. lipolytica YALI0C10054g (ALK11) and Y. lipolytica YALI0A20130g (ALK12).
67. The recombinant microorganism of any one of embodiments 55-66, wherein the fatty acyl desaturase is selected from an Argyrotaenia velutinana, Spodoptera litura, Sesamia inferens, Manduca sexta, Ostrinia nubilalis, Helicoverpa zea, Choristoneura rosaceana, Drosophila melanogaster, Spodoptera littoralis, Lampronia capitella, Amyelois transitella, Trichoplusia ni, Agrotis segetum, Ostrinia furnicalis, and Thalassiosira pseudonana derived fatty acyl desaturase.
68. The recombinant microorganism of any one of embodiments 55-67, wherein the fatty acyl desaturase has at least 95% sequence identity to a fatty acyl desaturase selected from the group consisting of: SEQ ID NOs: 39, 49-54, 58-63, 78-80 and GenBank Accession nos. AF416738, AGH12217.1, AI121943.1, CAJ43430.2, AF441221, AAF81787.1, AF545481, AJ271414, AY362879, ABX71630.1, NP001299594.1, Q9N9Z8, ABX71630.1 and AIM40221.1.
69. The recombinant microorganism of any one of embodiments 55-68, wherein the acyl-CoA oxidase is selected from Table 5a.
70. The recombinant microorganism of any one of embodiments 55-69, wherein the fatty alcohol forming fatty acyl reductase is selected from an Agrotis segetum, Spodoptera exigua, Spodoptera littoralis, Euglena gracilis, Yponomeuta evonymellus and Helicoverpa armigera derived fatty alcohol forming fatty acyl reductase.
71. The recombinant microorganism of any one of embodiments 55-70, wherein the fatty alcohol forming fatty acyl reductase has at least 95% sequence identity to a fatty alcohol forming fatty acyl reductase selected from the group consisting of: SEQ ID NOs: 1-3, 32, 41-48, 55-57, 73, 75, 77 and 82.
72. The recombinant microorganism of any one of embodiments 55-71, wherein the fatty acyl desaturase catalyzes the conversion of a fatty acyl-CoA into a mono- or poly-unsaturated intermediate selected from E5-10:Acyl-CoA, E7-12:Acyl-CoA, E9-14:Acyl-CoA, E11-16:Acyl-CoA, E13-18:Acyl-CoA, Z7-12:Acyl-CoA, Z9-14:Acyl-CoA, Z11-16:Acyl-CoA, Z13-18:Acyl-CoA, Z8-12:Acyl-CoA, Z10-14:Acyl-CoA, Z12-16:Acyl-CoA, Z14-18:Acyl-CoA, Z7-10:Acyl-coA, Z9-12:Acyl-CoA, Z11-14:Acyl-CoA, Z13-16:Acyl-CoA, Z15-18:Acyl-CoA, E7-10:Acyl-CoA, E9-12:Acyl-CoA, E11-14:Acyl-CoA, E13-16:Acyl-CoA, E15-18:Acyl-CoA, E5Z7-12:Acyl-CoA, E7Z9-12:Acyl-CoA, E9Z11-14:Acyl-CoA, E11Z13-16:Acyl-CoA, E13Z15-18:Acyl-CoA, E6E8-10:Acyl-CoA, E8E10-12:Acyl-CoA, E10E12-14:Acyl-CoA, E12E14-16:Acyl-CoA, Z5E8-10:Acyl-CoA, Z7E10-12:Acyl-CoA, Z9E12-14:Acyl-CoA, Z11E14-16:Acyl-CoA, Z13E16-18:Acyl-CoA, Z3-10:Acyl-CoA, Z5-12:Acyl-CoA, Z7-14:Acyl-CoA, Z9-16:Acyl-CoA, Z11-18:Acyl-CoA, Z3Z5-10:Acyl-CoA, Z5Z7-12:Acyl-CoA, Z7Z9-14:Acyl-CoA, Z9Z11-16:Acyl-CoA, Z11Z13-16:Acyl-CoA, and Z13Z15-18:Acyl-CoA.
73. The recombinant microorganism of any one of embodiments 55-72, wherein the mono- or poly-unsaturated ≤C18 fatty alcohol is selected from the group consisting of E5-10:OH, Z8-12:OH, Z9-12:OH, Z11-14:OH, Z11-16:OH, E11-14:OH, E8E10-12:OH, E7Z9-12:OH, Z11Z13-16OH, Z9-14:OH, Z9-16:OH, and Z13-18:OH.
74. The recombinant microorganism of any one of embodiments 55-73, wherein the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an aldehyde forming fatty acyl-CoA reductase capable of catalyzing the conversion of the mono- or poly-unsaturated ≤C18 fatty alcohol into a corresponding ≤C18 fatty aldehyde.
75. The recombinant microorganism of embodiment 74, wherein the aldehyde forming fatty acyl-CoA reductase is selected from the group consisting of Acinetobacter calcoaceticus A0A1C4HN78, A. calcoaceticus N9DA85, A. calcoaceticus R8XW24, A. calcoaceticus A0A1A0GGM5, A. calcoaceticus A0A117N158, and Nostoc punctiforme YP_001865324.
76. The recombinant microorganism of any one of embodiments 55-75, wherein the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an alcohol oxidase or an alcohol dehydrogenase capable of catalyzing the conversion of the mono- or poly-unsaturated ≤C18 fatty alcohol into a corresponding ≤C18 fatty aldehyde.
77. The recombinant microorganism of any one of embodiments 55-76, wherein the ≤C18 fatty aldehyde is selected from the group consisting of Z9-16:Ald, Z11-16:Ald, Z11Z13-16:Ald, and Z13-18:Ald.
78. The recombinant microorganism of any one of embodiments 55-77, wherein the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an acetyl transferase capable of catalyzing the conversion of the mono- or poly-unsaturated ≤C18 fatty alcohol into a corresponding ≤C18 fatty acetate.
79. The recombinant microorganism of embodiment 78, wherein the acetyl transferase is selected from Table 5d.
80. The recombinant microorganism of embodiment 78, wherein the ≤C18 fatty acetate is selected from the group consisting of E5-10:Ac, Z7-12:Ac, Z8-12:Ac, Z9-12:Ac, E7Z9-12:Ac, Z9-14:Ac, Z9E12-14:Ac, Z11-14:Ac, E11-14:Ac, Z9-16:Ac, and Z11-16:Ac.
81. The recombinant microorganism of any one of embodiments 55-80, wherein the recombinant microorganism further comprises:
-
- at least one endogenous or exogenous nucleic acid molecule encoding an enzyme selected from an alcohol oxidase, an alcohol dehydrogenase, and an aldehyde forming fatty acyl-CoA reductase capable of catalyzing the conversion of the mono- or poly-unsaturated ≤C18 fatty alcohol into a corresponding ≤C18 fatty aldehyde; and
- at least one endogenous or exogenous nucleic acid molecule encoding an acetyl transferase capable of catalyzing the conversion of the mono- or poly-unsaturated ≤C18 fatty alcohol into a corresponding ≤C18 fatty acetate.
82. The recombinant microorganism of embodiment 81, wherein the mono- or poly-unsaturated ≤C18 fatty aldehyde and ≤C18 fatty acetate is selected from the group consisting of E5-10:Ac, Z7-12:Ac, Z8-12:Ac, Z9-12:Ac, E7Z9-12:Ac, Z9-14:Ac, Z9E12-14:Ac, E11-14:Ac, Z11-14:Ac, Z11-16:Ac, Z9-16:Ac, Z9-16:Ald, Z11-16:Ald, Z11Z13-16:Ald, and Z13-18:Ald.
83. The recombinant microorganism of any one of embodiments 55-82, wherein the recombinant microorganism is a yeast selected from the group consisting of Yarrowia lipolytica, Saccharomyces cerevisiae, Candida albicans, Candida tropicalis and Candida viswanathii.
84. A method of producing a mono- or poly-unsaturated ≤C18 fatty alcohol from an endogenous or exogenous source of saturated C6-C24 fatty acid, comprising: cultivating the recombinant microorganism of any one of embodiment 55-83 in a culture medium containing a feedstock that provides a carbon source adequate for the production of the mono- or poly-unsaturated ≤C18 fatty alcohol.
85. The method of embodiment 84, wherein the mono- or poly-unsaturated ≤C18 fatty alcohol is selected from the group consisting of E5-10:OH, Z8-12:OH, Z9-12:OH, Z11-14:OH, Z11-16:OH, E11-14:OH, E8E10-12:OH, E7Z9-12:OH, Z11Z13-16OH, Z9-14:OH, Z9-16:OH, and Z13-18:OH.
86. The method of any one of embodiments 84-85, further comprising a step of recovering the mono- or poly-unsaturated ≤C18 fatty alcohol.
87. The method of embodiment 86, wherein said recovery step comprises distillation.
88. The method of embodiment 86, wherein said recovery step comprises membrane-based separation.
89. A method of engineering a microorganism that is capable of producing a mono- or poly-unsaturated ≤C18 fatty alcohol from an endogenous or exogenous source of saturated C6-C24 fatty acid, wherein the method comprises introducing into a microorganism the following:
-
- (a) at least one exogenous nucleic acid molecule encoding a fatty acyl desaturase that catalyzes the conversion of a saturated C6-C24 fatty acyl-CoA to a corresponding mono- or poly-unsaturated C6-C24 fatty acyl-CoA;
- (b) at least one exogenous nucleic acid molecule encoding an acyl-CoA oxidase that catalyzes the conversion of the mono- or poly-unsaturated C6-C24 fatty acyl-CoA from (a) into a mono- or poly-unsaturated ≤C18 fatty acyl-CoA after one or more successive cycle of acyl-CoA oxidase activity, with a given cycle producing a mono- or poly-unsaturated C4-C22 fatty acyl-CoA intermediate with a two carbon truncation relative to a starting mono- or poly-unsaturated C6-C24 fatty acyl-CoA substrate in that cycle; and
- (c) at least one exogenous nucleic acid molecule encoding a fatty alcohol forming fatty acyl reductase that catalyzes the conversion of the mono- or poly-unsaturated ≤C18 fatty acyl-CoA from (b) into the corresponding mono- or poly-unsaturated ≤C18 fatty alcohol.
90. The method of embodiment 89, wherein the microorganism is MATA ura3-302::SUC2 Δpox1 Δpox2 Δpox3 Δpox4 Δpox5 Δpox6 Δfadh Δadh1 Δadh2 Δadh3 Δadh4 Δadh5 Δadh6 Δadh7 Δfao1::URA3.
91. The method of any one of embodiments 89-90, wherein the method further comprises introducing into the microorganism at least one endogenous or exogenous nucleic acid molecule encoding an acyltransferase that preferably stores ≤C18 fatty acyl-CoA.
92. The method of any one of embodiments 89-91, wherein the method further comprises introducing into the microorganism at least one endogenous or exogenous nucleic acid molecule encoding an acyltransferase that preferably stores ≤C18 fatty acyl-CoA, and wherein the acyltransferase is selected from the group consisting of glycerol-3-phosphate acyl transferase (GPAT), lysophosphatidic acid acyltransferase (LPAAT), glycerolphospholipid acyltransferase (GPLAT) and diacylglycerol acyltransferases (DGAT).
93. The method of any one of embodiments 89-92, wherein the method further comprises introducing into the microorganism at least one endogenous or exogenous nucleic acid molecule encoding an acyltransferase that preferably stores ≤C18 fatty acyl-CoA, and wherein the acyltransferase is selected from Table 5b.
94. The method of any one of embodiments 89-93, wherein the method further comprises introducing into the microorganism at least one endogenous or exogenous nucleic acid molecule encoding an acylglycerol lipase that preferably hydrolyzes ester bonds of >C16, of >C14, of >C12 or of >C10 acylglycerol substrates.
95. The method of any one of embodiments 89-94, wherein the method further comprises introducing into the microorganism at least one endogenous or exogenous nucleic acid molecule encoding an acylglycerol lipase that preferably hydrolyzes ester bonds of >C16, of >C14, of >C12 or of >C10 acylglycerol substrates, and wherein the acylglycerol lipase is selected from Table 5c.
96. The method of any one of embodiments 89-95, wherein the method further comprises introducing into the microorganism one or more modifications comprising a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous enzyme that catalyzes a reaction in a pathway that competes with the biosynthesis pathway for the production of a mono- or poly-unsaturated ≤C18 fatty alcohol.
97. The method of any one of embodiments 89-96, wherein the method further comprises introducing into the microorganism one or more modifications comprising a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous enzyme selected from:
-
- (i) one or more acyl-CoA oxidase;
- (ii) one or more acyltransferase;
- (iii) one or more acylglycerol lipase and/or sterol ester esterase;
- (iv) one or more (fatty) alcohol dehydrogenase;
- (v) one or more (fatty) alcohol oxidase; and
- (vi) one or more cytochrome P450 monooxygenase.
98. The method of any one of embodiments 89-97, wherein the method further comprises introducing into the microorganism one or more modifications comprising a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous acyl-CoA oxidase enzyme selected from the group consisting of Y. lipolytica POX1(YALI0E32835g), Y. lipolytica POX2(YALI0F10857g), Y. lipolytica POX3(YALI0D24750g), Y. lipolytica POX4(YALI0E27654g), Y. lipolytica POX5(YALI0C23859g), Y. lipolytica POX6(YALI0E06567g); S. cerevisiae POX1(YGL205W); Candida POX2 (CaO19.1655, CaO19.9224, CTRG_02374, M18259), Candida POX4 (CaO19.1652, CaO19.9221, CTRG_02377, M12160), and Candida POX5 (CaO19.5723, CaO19.13146, CTRG_02721, M12161).
99. The method of any one of embodiments 89-98, wherein the method further comprises introducing into the microorganism one or more modifications comprising a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous acyltransferase enzyme selected from the group consisting of Y. lipolytica YALI0C00209g, Y. lipolytica YALI0E18964g, Y. lipolytica YALI0F19514g, Y. lipolytica YALI0C14014g, Y. lipolytica YALI0E16797g, Y. lipolytica YALI0E32769g, and Y. lipolytica YALI0D07986g, S. cerevisiae YBL011w, S. cerevisiae YDL052c, S. cerevisiae YOR175C, S. cerevisiae YPR139C, S. cerevisiae YNR008w, and S. cerevisiae YOR245c, and Candida I503_02577, Candida CTRG_02630, Candida CaO19.250, Candida CaO19.7881, Candida CTRG_02437, Candida CaO19.1881, Candida CaO19.9437, Candida CTRG_01687, Candida CaO19.1043, Candida CaO19.8645, Candida CTRG_04750, Candida CaO19.13439, Candida CTRG_04390, Candida CaO19.6941, Candida CaO19.14203, and Candida CTRG_06209.
100. The method of any one of embodiments 89-99, wherein the method further comprises introducing into the microorganism one or more modifications comprising a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous acylglycerol lipase and/or sterol ester esterase enzyme selected from the group consisting of Y. lipolytica YALI0E32035g, Y. lipolytica YALI0D17534g, Y. lipolytica YALI0F10010g, Y. lipolytica YALI0C14520g, and Y. lipolytica YALI0E00528g, S. cerevisiae YKL140w, S. cerevisiae YMR313c, S. cerevisiae YKR089c, S. cerevisiae YOR081c, S. cerevisiae YKL094W, S. cerevisiae YLL012W, and S. cerevisiae YLR020C, and Candida CaO19.2050, Candida CaO19.9598, Candida CTRG_01138, Candida W5Q_03398, Candida CTRG_00057, Candida CaO19.5426, Candida CaO19.12881, Candida CTRG_06185, Candida CaO19.4864, Candida CaO19.12328, Candida CTRG_03360, Candida CaO19.6501, Candida CaO19.13854, Candida CTRG_05049, Candida CaO19.1887, Candida CaO19.9443, Candida CTRG_01683, and Candida CTRG_04630.
101. The method of any one of embodiments 89-100, wherein the method further comprises one or more modifications comprising a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous cytochrome P450 monooxygenases selected from the group consisting of Y. lipolytica YALI0E25982g (ALK1), Y. lipolytica YALI0F01320g (ALK2), Y. lipolytica YALI0E23474g (ALK3), Y. lipolytica YALI0B13816g (ALK4), Y. lipolytica YALI0B13838g (ALK5), Y. lipolytica YALI0B01848g (ALK6), Y. lipolytica YALI0A15488g (ALK7), Y. lipolytica YALI0C12122g (ALK8), Y. lipolytica YALI0B06248g (ALK9), Y. lipolytica YALI0B20702g (ALK10), Y. lipolytica YALI0C10054g (ALK11) and Y. lipolytica YALI0A20130g (ALK12).
102. The method of any one of embodiments 89-101, wherein the fatty acyl desaturase is selected from an Argyrotaenia velutinana, Spodoptera litura, Sesamia inferens, Manduca sexta, Ostrinia nubilalis, Helicoverpa zea, Choristoneura rosaceana, Drosophila melanogaster, Spodoptera littoralis, Lampronia capitella, Amyelois transitella, Trichoplusia ni, Agrotis segetum, Ostrinia furnicalis, and Thalassiosira pseudonana derived fatty acyl desaturase.
103. The method of any one of embodiments 89-102, wherein the fatty acyl desaturase has at least 95% sequence identity to a fatty acyl desaturase selected from the group consisting of: SEQ ID NOs: 39, 49-54, 58-63, and GenBank Accession nos. AF416738, AGH12217.1, AII21943.1, CAJ43430.2, AF441221, AAF81787.1, AF545481, AJ271414, AY362879, ABX71630.1, NP001299594.1, Q9N9Z8, ABX71630.1 and AIM40221.1.
104. The method of any one of embodiments 89-103, wherein the acyl-CoA oxidase is selected from Table 5a.
105. The method of any one of embodiments 89-104, wherein the fatty alcohol forming fatty acyl reductase is selected from an Agrotis segetum, Spodoptera exigua, Spodoptera littoralis, Euglena gracilis, Yponomeuta evonymellus and Helicoverpa armigera derived fatty alcohol forming fatty acyl reductase.
106. The method of any one of embodiments 89-105, wherein the fatty alcohol forming fatty acyl reductase has at least 90% sequence identity to a fatty alcohol forming fatty acyl reductase selected from the group consisting of: SEQ ID NOs: 1-3, 32, 41-48, 55-57, 73, 75, 77 and 82.
107. The method of any one of embodiments 89-106, wherein the fatty acyl desaturase catalyzes the conversion of a fatty acyl-CoA into a mono- or poly-unsaturated intermediate selected from E5-10:Acyl-CoA, E7-12:Acyl-CoA, E9-14:Acyl-CoA, E11-16:Acyl-CoA, E13-18:Acyl-CoA, Z7-12:Acyl-CoA, Z9-14:Acyl-CoA, Z11-16:Acyl-CoA, Z13-18:Acyl-CoA, Z8-12:Acyl-CoA, Z10-14:Acyl-CoA, Z12-16:Acyl-CoA, Z14-18:Acyl-CoA, Z7-10:Acyl-coA, Z9-12:Acyl-CoA, Z11-14:Acyl-CoA, Z13-16:Acyl-CoA, Z15-18:Acyl-CoA, E7-10:Acyl-CoA, E9-12:Acyl-CoA, E11-14:Acyl-CoA, E13-16:Acyl-CoA, E15-18:Acyl-CoA, E5Z7-12:Acyl-CoA, E7Z9-12:Acyl-CoA, E9Z11-14:Acyl-CoA, E11Z13-16:Acyl-CoA, E13Z15-18:Acyl-CoA, E6E8-10:Acyl-CoA, E8E10-12:Acyl-CoA, E10E12-14:Acyl-CoA, E12E14-16:Acyl-CoA, Z5E8-10:Acyl-CoA, Z7E10-12:Acyl-CoA, Z9E12-14:Acyl-CoA, Z11E14-16:Acyl-CoA, Z13E16-18:Acyl-CoA, Z3-10:Acyl-CoA, Z5-12:Acyl-CoA, Z7-14:Acyl-CoA, Z9-16:Acyl-CoA, Z11-18:Acyl-CoA, Z3Z5-10:Acyl-CoA, Z5Z7-12:Acyl-CoA, Z7Z9-14:Acyl-CoA, Z9Z11-16:Acyl-CoA, Z11Z13-16:Acyl-CoA, and Z13Z15-18:Acyl-CoA.
108. The method of any one of embodiments 89-107, wherein the mono- or poly-unsaturated ≤C18 fatty alcohol is selected from the group consisting of E5-10:OH, Z8-12:OH, Z9-12:OH, Z11-14:OH, Z11-16:OH, E11-14:OH, E8E10-12:OH, E7Z9-12:OH, Z11Z13-16OH, Z9-14:OH, Z9-16:OH, and Z13-18:OH.
109. The method of any one of embodiments 89-108, wherein the method further comprises introducing into the microorganism at least one endogenous or exogenous nucleic acid molecule encoding an aldehyde forming fatty acyl-CoA reductase capable of catalyzing the conversion of the mono- or poly-unsaturated ≤C18 fatty alcohol into a corresponding ≤C18 fatty aldehyde.
110. The method of embodiment 109, wherein the aldehyde forming fatty acyl-CoA reductase is selected from the group consisting of Acinetobacter calcoaceticus A0A1C4HN78, A. calcoaceticus N9DA85, A. calcoaceticus R8XW24, A. calcoaceticus A0A1A0GGM5, A. calcoaceticus A0A117N158, and Nostoc punctiforme YP_001865324.
111. The method of any one of embodiments 89-110, wherein the method further comprises introducing into the microorganism at least one endogenous or exogenous nucleic acid molecule encoding an alcohol oxidase or an alcohol dehydrogenase capable of catalyzing the conversion of the mono- or poly-unsaturated ≤C18 fatty alcohol into a corresponding ≤C18 fatty aldehyde.
112. The method of any one of embodiments 109-111, wherein the ≤C18 fatty aldehyde is selected from the group consisting of Z9-16:Ald, Z11-16:Ald, Z11Z13-16:Ald, and Z13-18:Ald.
113. The method of any one of embodiments 89-112, wherein method further comprises introducing into the microorganism at least one endogenous or exogenous nucleic acid molecule encoding an acetyl transferase capable of catalyzing the conversion of the mono- or poly-unsaturated ≤C18 fatty alcohol into a corresponding ≤C18 fatty acetate.
114. The method of embodiment 113, wherein the acetyl transferase is selected from Table 5d.
115. The method of any one of embodiment 113-114, wherein the ≤C18 fatty acetate is selected from the group consisting of E5-10:Ac, Z7-12:Ac, Z8-12:Ac, Z9-12:Ac, E7Z9-12:Ac, Z9-14:Ac, Z9E12-14:Ac, E11-14:Ac, Z9-16:Ac, Z11-14:Ac and Z11-16:Ac.
116. The method of any one of embodiments 89-115, wherein the method further comprises introducing into the microorganism:
-
- at least one endogenous or exogenous nucleic acid molecule encoding an enzyme selected from an alcohol oxidase, an alcohol dehydrogenase, and an aldehyde forming fatty acyl-CoA reductase capable of catalyzing the conversion of the mono- or poly-unsaturated ≤C18 fatty alcohol into a corresponding ≤C18 fatty aldehyde; and
- at least one endogenous or exogenous nucleic acid molecule encoding an acetyl transferase capable of catalyzing the conversion of the mono- or poly-unsaturated ≤C18 fatty alcohol into a corresponding ≤C18 fatty acetate.
117. The method of embodiment 116, wherein the mono- or poly-unsaturated ≤C18 fatty aldehyde and ≤C18 fatty acetate is selected from the group consisting of E5-10:Ac, Z7-12:Ac, Z8-12:Ac, Z9-12:Ac, E7Z9-12:Ac, Z9-14:Ac, Z9E12-14:Ac, Z11-14:Ac, E11-14:Ac, Z11-16:Ac, Z9-16:Ald, Z9-16:Ac, Z11-16:Ald, Z11Z13-16:Ald, and Z13-18:Ald.
118. A method of producing a mono- or poly-unsaturated ≤C18 fatty aldehyde from an endogenous or exogenous source of saturated C6-C24 fatty acid, comprising: cultivating the recombinant microorganism of any one of embodiments 74-76 in a culture medium containing a feedstock that provides a carbon source adequate for the production of the mono- or poly-unsaturated ≤C18 fatty aldehyde.
119. The method of embodiment 118, wherein the ≤C18 fatty aldehyde is selected from the group consisting of Z9-16:Ald, Z11-16:Ald, Z11Z13-16:Ald, and Z13-18:Ald.
120. The method of any one of embodiments 118-119, further comprising a step of recovering the mono- or poly-unsaturated ≤C18 fatty aldehyde.
121. The method of embodiment 120, wherein said recovery step comprises distillation.
122. The method of embodiment 120, wherein said recovery step comprises membrane-based separation.
123. A method of producing a mono- or poly-unsaturated ≤C18 fatty acetate from an endogenous or exogenous source of saturated C6-C24 fatty acid, comprising: cultivating the recombinant microorganism of any one of embodiments 78-80 in a culture medium containing a feedstock that provides a carbon source adequate for the production of the mono- or poly-unsaturated ≤C18 fatty acetate.
124. The method of embodiment 123, wherein the mono- or poly-unsaturated ≤C18 fatty acetate is selected from the group consisting of E5-10:Ac, Z7-12:Ac, Z8-12:Ac, Z9-12:Ac, E7Z9-12:Ac, Z9-14:Ac, Z9E12-14:Ac, Z11-14:Ac, E11-14:Ac, Z9-16:Ac, and Z11-16:Ac.
125. The method of any one of embodiments 123-124, further comprising a step of recovering the mono- or poly-unsaturated ≤C18 fatty acetate.
126. The method of embodiment 125, wherein said recovery step comprises distillation.
127. The method of embodiment 125, wherein said recovery step comprises membrane-based separation.
128. The method of any one of embodiments 89-115, wherein the recombinant microorganism is a yeast selected from the group consisting of Yarrowia lipolytica, Saccharomyces cerevisiae, Candida albicans, Candida viswanathii and Candida tropicalis.
129. The method of any one of embodiments 89-115, wherein the mono- or poly-unsaturated ≤C18 fatty alcohol is converted into a corresponding ≤C18 fatty aldehyde using chemical methods.
130. The method of embodiment 129, wherein the chemical methods are selected from TEMPO-bleach, TEMPO-copper-air, TEMPO-PhI(OAc)2, Swern oxidation and noble metal-air.
131. The method of any one of embodiments 89-115, wherein the mono- or poly-unsaturated ≤C18 fatty alcohol is converted into a corresponding ≤C18 fatty acetate using chemical methods.
132. The method of embodiment 131, wherein the chemical methods utilize a chemical agent selected from the group consisting of acetyl chloride, acetic anhydride, butyryl chloride, butyric anhydride, propanoyl chloride and propionic anhydride in the presence of 4-N,N-dimethylaminopyridine (DMAP) or sodium acetate to esterify the mono- or poly-unsaturated ≤C18 fatty alcohol to the corresponding ≤C18 fatty acetate.
Claims
1. A method of producing a C6-C24 fatty alcohol, said method comprising: wherein the C6-C24 fatty acyl-CoA is converted to a C6-C24 fatty alcohol by catalytic activity of the fatty alcohol-forming fatty acyl reductase.
- a) providing a recombinant Yarrowia lipolytica microorganism comprising a heterologous nucleic acid molecule encoding and expressing a Euglena fatty alcohol-forming fatty acyl reductase that catalyzes conversion of C6-C24 fatty acyl-CoA into a corresponding mono- or poly-unsaturated C6-C24 fatty alcohol; and
- b) cultivating the recombinant Yarrowia lipolytica of (a) in a culture medium containing a carbon source feedstock and C6-C24 fatty acyl-CoA;
2. The method of claim 1, wherein the Euglena fatty alcohol-forming fatty acyl reductase is from Euglena gracilis.
3. The method of claim 1, wherein the C6-C24 fatty alcohol is selected from the group consisting of (Z)-3-hexen-1-ol, (Z)-3-nonen-1-ol, (E)-2-decen-1-ol, (Z)-2-decen-1-ol, (E)-3-decen-1-ol, (Z)-3-decen-1-ol, (Z)-4-decen-1-ol, (E)-5-decen-1-ol, (Z)-5-decen-1-ol, (E)-8-decen-1-ol, (Z,Z)-4,7-decadien-1-ol, (Z)-3-dodecen-1-ol, (E)-5-dodecen-1-ol, (Z)-5-dodecen-1-ol, (E)-6-dodecen-1-ol, (E)-7-dodecen-1-ol, (Z)-7-dodecen-1-ol, (E)-8-dodecen-1-ol, (Z)-8-dodecen-1-ol, (E)-9-dodecen-1-ol, (Z)-9-dodecen-1-ol, (E)-10-dodecen-1-ol, (Z)-10-dodecen-1-ol, (Z,Z)-3,6-dodecadien-1-ol, (E,E)-5,7-dodecadien-1-ol, (E,Z)-5,7-dodecadien-1-ol, (Z,E)-5,7-dodecadien-1-ol, (E,Z)-7,9-dodecadien-1-ol, (Z,E)-7,9-dodecadien-1-ol, (Z,Z)-7,9-dodecadien-1-ol, (E,E)-8,10-dodecadien-1-ol, (E,Z)-8,10-dodecadien-1-ol, (Z,E)-8,10-dodecadien-1-ol, (Z,Z)-8,10-dodecadien-1-ol, (Z,E,E)-3,6,8-dodecatrien-1-ol, (Z,Z,E)-3,6,8-dodecatrien-1-ol, (Z,Z)-4,7-tridecadien-1-ol, (E)-3-tetradecen-1-ol, (Z)-3-tetradecen-1-ol, (E)-5-tetradecen-1-ol, (Z)-5-tetradecen-1-ol, (E)-7-tetradecen-1-ol, (Z)-7-tetradecen-1-ol, (Z)-8-tetradecen-1-ol, (E)-9-tetradecen-1-ol, (Z)-9-tetradecen-1-ol, (E)-11-tetradecen-1-ol, (Z)-11-tetradecen-1-ol, (Z,Z)-5,8-tetradecadien-1-ol, (E,E)-8,10-tetradecadien-1-ol, (Z,E)-8,10-tetradecadien-1-ol, (Z,E)-9,11-tetradecadien-1-ol, (Z,Z)-9,11-tetradecadien-1-ol, (Z,E)-9,12-tetradecadien-1-ol, (Z,Z)-9,12-tetradecadien-1-ol, (E,E)-10,12-tetradecadien-1-ol, (Z,Z)-10,12-tetradecadien-1-ol, (E)-8-pentadecen-1-ol, (Z)-8-pentadecen-1-ol, (Z,Z)-6,9-pentadecadien-1-ol, (E,Z)-8,10-pentadecadien-1-ol, (E)-5-hexadecen-1-ol, (Z)-5-hexadecen-1-ol, (E)-7-hexadecen-1-ol, (Z)-7-hexadecen-1-ol, (E)-9-hexadecen-1-ol, (Z)-9-hexadecen-1-ol, (E)-10-hexadecen-1-ol, (E)-11-hexadecen-1-ol, (Z)-11-hexadecen-1-ol, (E,E)-1,3-hexadecadien-1-ol, (E,Z)-4,6-hexadecadien-1-ol, (Z,Z)-7,10-hexadecadien-1-ol, (Z,E)-7,11-hexadecadien-1-ol, (Z,Z)-7,11-hexadecadien-1-ol, (E,E)-10,12-hexadecadien-1-ol, (E,Z)-10,12-hexadecadien-1-ol, (E,E)-11,13-hexadecadien-1-ol, (E,Z)-11,13-hexadecadien-1-ol, (Z,E)-11,13-hexadecadien-1-ol, (Z,Z)-11,13-hexadecadien-1-ol, (E,E,Z)-4,6,10-hexadecatrien-1-ol, (E,Z,Z)-4,6,10-hexadecatrien-1-ol, (E)-8-heptadecen-1-ol, (Z)-8-heptadecen-1-ol, (Z)-11-heptadecen-1-ol, (Z,Z)-8,10-heptadecadien-1-ol, (E)-9-octadecen-1-ol, (Z)-9-octadecen-1-ol, (E)-11-octadecen-1-ol, (Z)-11-octadecen-1-ol, (Z)-13-octadecen-1-ol, (E,Z)-2,13-octadecadien-1-ol, (Z,Z)-2,13-octadecadien-1-ol, (E,E)-5,9-octadecadien-1-ol, (E,E)-9,12-octadecadien-1-ol, (E,E,E)-9,12,15-octadecatrien-1-ol, and combinations thereof.
4. The method of claim 1, wherein the recombinant Yarrowia lipolytica microorganism comprises a nucleic acid molecule encoding an alcohol oxidase or an alcohol dehydrogenase that catalyzes the conversion of the C6-C24 fatty alcohol into a corresponding C6-C24 fatty aldehyde.
5. The method of claim 4, wherein the alcohol dehydrogenase is selected from the group consisting of Q9NAR7, P14940, Q00669, P21518, P25139, P48584, P22245, Q9NG42, P48585, Q09009, P51549, P21898, Q07588, Q9NG40, P10807, P07162, Q09010, Q00671, Q00672, P07159, P84328, P23361, P23277, Q9U8S9, P23278, Q03384, P28484, P51550, P17648, P48977, P25988, Q00670, P50381, P51552, P42327, P25721, P00334, Q6LCE4, Q24641, P26719, Q50L96, P28483, P51551, Q05114, P37473, Q9GN94, Q0KDL6, P9WQC2, Q8NXU1, P9WQC3, Q7A742, Q2YSX0, Q4J781, Q5HI63, Q2G0G1, Q6GJ63, Q6GBM4, Q2FJ31, Q99W07, Q8CQ56, Q5HRD6, P39462, Q96XE0, P30350, P49645, P48814, Q70UN9, P23991, P86883, P19631, P23236, P48586, P22246, P07161, P12854, Q9Z2M2, Q64413, Q64415, P05336, Q07288, P00333, P86885, P14219, P25141, Q03505, P14673, P80338, P13603, Q07264, P00330, P06525, P00332, Q27404, P00329, P41680, P06757, B4M8Y0, O00097, P41747, Q9P6C8, P43067, Q2R8Z5, P09370, P20369, Q75ZX4, Q5RBP7, P07327, P28469, Q5R1W2, P14139, P00325, P00326, O97959, P48815, Q70UP5, Q70UP6, P27581, P25720, P23237, P48587, P07160, P24267, Q24803, P10847, Q9P4C2, P04707, Q4R1E8, P28032, P14674, O13309, Q96533, P00331, O94038, Q0ITW7, P09369, P49383, O46649, O46650, P10848, P14675, P49384, P07246, P08319, Q9QYY9, Q64563, P80468, P49385, Q09669, A6ZTT5, P10127, Q6XQ67, Q3ZC42, P19854, P28474, P12711, O19053, P11766, Q54TC2, P38113, P28332, P41681, Q5R7Z8, Q5XI95, P40394, P41682, Q64437, P9WQC0, O31186, P9WQC1, P20368, Q7U1B9, P9WQC6, P9WQC7, P0DJA2, F8DVL8, P9WQB8, P9WQB9, P33744, P0A9Q7, P0A9Q8, P72324, A2XAZ3, P39451, P12311, P08843, P54202, P07754, Q9SK86, Q9SK87, A1L4Y2, Q8VZ49, Q0V7W6, Q8LEB2, Q9FH04, P93629, P46415, P71017, Q17335, Q0DWH1, O07737, Q17334, O45687, P33010, P37686, P81786, P80222, P85440, P26325, P80512, P12886, P22797, P25405, P25406, P00327, P00328, P42328, P81600, P81601, P80360, P81431, P80572, P80467, P86884, P79896, A0A0F6Q2K7, A0A0F6Q2W6, AKD01723.1, A0A0F6Q4H2, A0A0F6Q1E8, A0A0F6Q2K3, A0A0F6Q4H7, A0A0F6Q2J9, A0A0F6Q0W0, A0A0F6Q0V0, A0A0F6Q1F1, A0A0F6Q2X2, A0A0F6Q4I2, A0A0F6Q2X0, A0A0F6Q2L9, A0A0F6Q4K1, A0A0F6Q4J7, A0A0F6Q2Y5, A0A0F6Q2Y1, A0A0F6Q1G6, A0A0F6Q2Y9, A0A0F6Q0X5, A0A0F6Q2M3, A0A0F6Q2L1, A0A0F6Q1F9, A0A0F6Q0W6, A0A0F6Q1G9, A0A0F6Q2L4, A0A0F6Q2X6, A0A0F6Q1H3, A0A0F6Q0X1, NP_001188510.1, XP_001655103.1, ETN64198.1, YALI0F09603g (FADH), YALI0D25630g (ADH1), YALI0E17787g (ADH2), YALI0A16379g (ADH3), YALI0E15818g (ADH4), YALI0D02167g (ADH5), YALI0A15147g (ADH6), and YALI0E07766g (ADH7).
6. The method of claim 4, wherein the recombinant Yarrowia lipolytica microorganism produces a C6-C24 fatty aldehyde selected from the group consisting of (E)-2-decenal, (Z)-2-decenal, (Z)-4-decenal, (Z)-5-decenal, (E,E)-2,4-decadienal, (E,Z)-2,4-decadienal, (Z,Z)-2,4-decadienal, (E)-2-undecenal, (E)-2-dodecenal, (Z)-5-dodecenal, (E)-6-dodecenal, (E)-7-dodecenal, (Z)-7-dodecenal, (E)-8-dodecenal, (E)-9-dodecenal, (Z)-9-dodecenal, (E)-10-dodecenal, (E,Z)-5,7-dodecadienal, (Z,E)-5,7-dodecadienal, (Z,Z)-5,7-dodecadienal, (E,Z)-7,9-dodecadienal, (E,E)-8,10-dodecadienal, (E,Z)-8,10-dodecadienal, (Z,E)-8,10-dodecadienal, (Z)-4-tridecenal, (E)-5-tetradecenal, (Z)-5-tetradecenal, (Z)-7-tetradecenal, (Z)-8-tetradecenal, (Z)-9-tetradecenal, (E)-11-tetradecenal, (Z)-11-tetradecenal, (E,E)-2,4-tetradecadienal, (E,Z)-4,9-tetradecadienal, (E,E)-5,8-tetradecadienal, (Z,Z)-5,8-tetradecadienal, (E,E)-8,10-tetradecadienal, (E,Z)-8,10-tetradecadienal, (Z,Z)-8,10-tetradecadienal, (Z,E)-9,11-tetradecadienal, (Z,Z)-9,11-tetradecadienal, (Z,E)-9,12-tetradecadienal, (E,E)-10,12-tetradecadienal, (Z)-10-pentadecenal, (Z,Z)-6,9-pentadecadienal, (E,Z)-9,11-pentadecadienal, (Z,Z)-9,11-pentadecadienal, (E)-7-hexadecenal, (Z)-7-hexadecenal, (E)-9-hexadecenal, (Z)-9-hexadecenal, (E)-10-hexadecenal, (Z)-10-hexadecenal, (E)-11-hexadecenal, (Z)-11-hexadecenal, (Z)-12-hexadecenal, (E)-14-hexadecenal, (E,Z)-4,6-hexadecadienal, (E,Z)-6,11-hexadecadienal, (Z,E)-7,11-hexadecadienal, (Z,Z)-7,11-hexadecadienal, (E,Z)-8,11-hexadecadienal, (E,E)-9,11-hexadecadienal, (E,Z)-9,11-hexadecadienal, (Z,E)-9,11-hexadecadienal, (Z,Z)-9,11-hexadecadienal, (E,E)-10,12-hexadecadienal, (E,Z)-10,12-hexadecadienal, (Z,E)-10,12-hexadecadienal, (Z,Z)-10,12-hexadecadienal, (E,E)-11,13-hexadecadienal, (E,Z)-11,13-hexadecadienal, (Z,E)-11,13-hexadecadienal, (Z,Z)-11,13-hexadecadienal, (E,E)-10,14-hexadecadienal, (E,E,Z)-4,6,11-hexadecatrienal, (Z,Z,E)-7,11,13-hexadecatrienal, (E,E,E)-10,12,14-hexadecatrienal, (E,E,Z)-10,12,14-hexadecatrienal, (E,E,Z,Z)-4,6,11,13-hexadecatetraenal, (E)-2-heptadecenal, (Z)-2-heptadecenal, (Z)-9-heptadecenal, (E)-2-octadecenal, (Z)-2-octadecenal, (E)-9-octadecenal, (Z)-9-octadecenal, (E)-11-octadecenal, (Z)-11-octadecenal, (E)-13-octadecenal, (Z)-13-octadecenal, (E)-14-octadecenal, (E,Z)-2,13-octadecadienal, (E,Z)-3,13-octadecadienal, (Z,Z)-3,13-octadecadienal, (Z,Z)-9,12-octadecadienal, (Z,Z)-11,13-octadecadienal, (E,E)-11,14-octadecadienal, (Z,Z)-13,15-octadecadienal, and (Z,Z, Z)-9,12,15-octadecatrienal.
7. The method of claim 1, wherein the recombinant Yarrowia lipolytica microorganism comprises a nucleic acid molecule encoding an acetyl transferase that catalyzes the conversion of the C6-C24 fatty alcohol into a corresponding C6-C24 fatty acetate.
8. The method of claim 7, wherein the acetyl transferase is selected from the group consisting of AY242066, AY242065, AY242064, AY242063, AY242062, EHJ65205, ACX53812, NP_001182381, EHJ65977, EHJ68573, KJ579226, GU594061, KTA99184.1, AIN34693.1, AY605053, XP_002552712.1, XP_503024.1, XP_505595.1, and XP_505513.1.
9. The method of claim 1, wherein the recombinant Yarrowia lipolytica microorganism comprises a nucleic acid molecule encoding an acyl transferase selected from the group consisting of AY242066, AY242065, AY242064, AY242063, AY242062, EHJ65205, ACX53812, NP_001182381, EHJ65977, EHJ68573, KJ579226, GU594061, KTA99184.1, AIN34693.1, AY605053, XP_002552712.1, XP_503024.1, XP_505595.1, XP_505513.1, AAL49962.1, BAC43739.1, AAH89846.1, F6TMU0, F6PXX7, F7B020, ALT83519.1, ANN46862.1, ANN46863.1, ANN46864.1, ANN46865.1, AAC49119.1, JAT48335.1, XP_008793203.1, XP_008806896.1, XP_008806740.1, XP_010908895.1, XP_010908896.1, Q96UY2, A0A077WEU5, A0A068SDP4, A0A068RXA2, A0A197JCE2, A0A1C7N060, I1BLC3, A0A1C7NC56, A0A077X3B5, Q96UY1, A0A077WVD4, A0A163K8G3, S2J8P3, A0A168J818, A0A0C9MR10, A0A162PN39, A0A167QXD0, A0A0C9M4C3, A0A0B7NDT1, A0A015LM78, A0A0B7NHQ3, A0A0A1NVK5, A0A0A1P436, A0A0D7BI48, A0A1B9HZT8, A0A1D1XN50, A0A1B9ILF0, S2JU94, A0A1B9GCB0, A0A068RKT0, Q5KFU4, Q55QC2, U5GY58, A0A197KA94, A0A088FR92, A0A194SBY3, E6R8N8, M7WKS9, A0A191UMW0, C6KZS6, J9VS50, A0A109FM23, I4YE91, A0A066WAJ3, A0A151VHJ4, A0A168LDJ3, A0A0A1ULK8, A0A074RWU7, A0A0K6FWT6, R9AL76, E6ZMU5, A0A0K3CJX4, A0A162Y103, A0A0B7FYU9, A0A1A5ZUI2, A0A1B9GXE9, V5EIP7, A0A127ZHG0, M5FTN9, A0A166HX72, A0A067QH80, A0A165PFB6, G7DXE4, A0A165KJK5, A0A0F7TLQ7, S8FI87, S7ZL04, I2FMX3, F8P370, V2WTH2, S7Q9H4, W3VTZ4, B8M0V7, A0A0D7B6H5, R7SCW4, A0A093UWD0, B6Q8Q9, A0A093VC12, A0A167SF58, A0A180GQ68, E3KWZ5, F4S978, A0A0U5GN87, W9WBT1, A0A0D2A9G0, S3DKQ1, A0A167S691, A0A0C3G1P8, A0A117NM34, A0A0M8NPT1, M2R3J5, A0A1E3JS60, V9DJY4, A0A1C1D128, A0A194XRZ1, A0A135LQY4, F2S034, A0A059J710, R7YTC1, A0A0G4PR11, F2SHG6, A0A022VWY8, A0A178F1Q9, A0A022XM67, F2PHM1, A0A178FDV0, A0A0F8UUV5, A0A0F8XD12, D8Q1Z6, A0A0L0VQ99, W6QE33, A0A0J0XU39, K2RIY7, A0A1B9HIE8, A0A0A2KLE4, A0A177FP94, Q0CU51, A0A0D2C195, K9GS70, K9H4T7, A0A0A2IRX2, A0A165XA55, A0A1E3HS30, R0JHT6, W6XT38, K1WNS8, A0A077R6Q5, A0A0G2F2K4, M2UB23, N4WZB4, A0A0D2ECJ4, K5ULK6, A0A081CNS6, W7E3D1, A0A0D1YAT0, V5FVB4, A0A150V2J4, A0A0D2P224, C5FY83, A0A0E9NND3, A0A163JYI7, M2SYN8, A0A0D2A9Y8, B2WFQ5, A0A178Z686, R1GYF1, A0A0D2AM77, A0A067TPJ7, A0A0G2DT71, A0A0S6XG57, A1CD57, W6ZE59, W9X299, A0A0L1HS74, E3RYE6, A0A178C491, A0A0D2JW30, A0A100ISZ7, G7XRR4, E4ZGH1, A0A0C3AU69, A0A0U1M481, A0A179UDB8, A0A177DML0, A0A074XTA2, R8BK00, A0A178E1M9, A0A074XCF2, A0A178CVL7, J4H349, F2T2H3, T5C9R0, C5GGF5, F8Q4F5, A0A074YHW3, A0A0D2E953, A0A0D2ETM7, A0A163ADJ9, U7PLY5, A0A0F2MF45, A0A0C2J820, A0A1E3B843, A0A0L6WTD3, G2YTS7, W9XGA9, A0A0F4YS69, M9LWR9, A0A074WDM7, M3CBZ0, A0A0C7C2J7, W9YU83, I8IUH8, A0A139HZI0, E9DGY4, A0A0J6F9P8, H6BM52, Q2UDX3, M3ASJ4, A0A177BZU0, A0A017S910, A0A175VVF2, A0A0J8UWI6, A0A0J6YFS7, J3K3F7, A0A0D2FX82, A0A072PSS5, A0A0A1MWE2, W2RSU8, C0S1D5, C1G9R2, A0A1D2JGH6, A0A166PXN0, Q54GC1, A0A0H1B9A9, R4XEF3, D3B2U8, U1HHT8, A0A1E3JYY5, A0A0C3JN41, B6HF05, A0A060S368, K5W449, B0CTA0, F0XD96, and A0A165EP91.
10. The method of claim 7, wherein the recombinant Yarrowia lipolytica microorganism produces a C6-C24 fatty acetate selected from the group consisting of (E)-2-decenyl acetate, (Z)-2-decenyl acetate, (Z)-3-decenyl acetate, (E)-4-decenyl acetate, (Z)-4-decenyl acetate, (E)-5-decenyl acetate, (Z)-5-decenyl acetate, (E)-7-decenyl acetate, (Z)-7-decenyl acetate, (E,E)-3,5-decadienyl acetate, (Z,E)-3,5-decadienyl acetate, (Z,Z)-4,7-decadienyl acetate, (E)-2-undecenyl acetate, (Z)-5-undecenyl acetate, (Z)-7-undecenyl acetate, (Z)-8-undecenyl acetate, (Z)-9-undecenyl acetate, (E)-3-dodecenyl acetate, (Z)-3-dodecenyl acetate, (E)-4-dodecenyl acetate, (E)-5-dodecenyl acetate, (Z)-5-dodecenyl acetate, (Z)-6-dodecenyl acetate, (E)-7-dodecenyl acetate, (Z)-7-dodecenyl acetate, (E)-8-dodecenyl acetate, (Z)-8-dodecenyl acetate, (E)-9-dodecenyl acetate, (Z)-9-dodecenyl acetate, (E)-10-dodecenyl acetate, (Z)-10-dodecenyl acetate, (E,Z)-3,5-dodecadienyl acetate, (Z,E)-3,5-dodecadienyl acetate, (E,E)-4,10-dodecadienyl acetate, (E,E)-5,7-dodecadienyl acetate, (E,Z)-5,7-dodecadienyl acetate, (Z,E)-5,7-dodecadienyl acetate, (Z,Z)-5,7-dodecadienyl acetate, (E,E)-7,9-dodecadienyl acetate, (E,Z)-7,9-dodecadienyl acetate, (Z,E)-7,9-dodecadienyl acetate, (Z,Z)-7,9-dodecadienyl acetate, (E,E)-8,10-dodecadienyl acetate, (E,Z)-8,10-dodecadienyl acetate, (Z,E)-8,10-dodecadienyl acetate, (Z,Z)-8,10-dodecadienyl acetate, (E)-2-tridecenyl acetate, (Z)-2-tridecenyl acetate, (E)-3-tridecenyl acetate, (E)-4-tridecenyl acetate, (Z)-4-tridecenyl acetate, (E)-6-tridecenyl acetate, (Z)-7-tridecenyl acetate, (E)-8-tridecenyl acetate, (Z)-8-tridecenyl acetate, (E)-9-tridecenyl acetate, (Z)-9-tridecenyl acetate, (Z)-10-tridecenyl acetate, (E)-11-tridecenyl acetate, (Z)-11-tridecenyl acetate, (E,Z)-4,7-tridecadienyl acetate, (Z,Z)-4,7-tridecadienyl acetate, (E,Z)-5,9-tridecadienyl acetate, (Z,E)-5,9-tridecadienyl acetate, (Z,Z)-5,9-tridecadienyl acetate, (Z,Z)-7,11-tridecadienyl acetate, (E,Z,Z)-4,7,10-tridecatrienyl acetate, (E)-3-tetradecenyl acetate, (Z)-3-tetradecenyl acetate, (E)-5-tetradecenyl acetate, (Z)-5-tetradecenyl acetate, (E)-6-tetradecenyl acetate, (Z)-6-tetradecenyl acetate, (E)-7-tetradecenyl acetate, (Z)-7-tetradecenyl acetate, (E)-8-tetradecenyl acetate, (Z)-8-tetradecenyl acetate, (E)-9-tetradecenyl acetate, (Z)-9-tetradecenyl acetate, (E)-10-tetradecenyl acetate, (Z)-10-tetradecenyl acetate, (E)-11-tetradecenyl acetate, (Z)-11-tetradecenyl acetate, (E)-12-tetradecenyl acetate, (Z)-12-tetradecenyl acetate, (E,E)-3,5-tetradecadienyl acetate, (E,Z)-3,5-tetradecadienyl acetate, (Z,E)-3,5-tetradecadienyl acetate, (E,Z)-3,7-tetradecadienyl acetate, (E,Z)-3,8-tetradecadienyl acetate, (E,Z)-4,9-tetradecadienyl acetate, (E,Z)-4,10-tetradecadienyl acetate, (Z,Z)-5,8-tetradecadienyl acetate, (E,E)-8,10-tetradecadienyl acetate, (E,Z)-8,10-tetradecadienyl acetate, (Z,E)-8,10-tetradecadienyl acetate, (E,E)-9,11-tetradecadienyl acetate, (E,Z)-9,11-tetradecadienyl acetate, (Z,E)-9,11-tetradecadienyl acetate, (Z,Z)-9,11-tetradecadienyl acetate, (E,E)-9,12-tetradecadienyl acetate, (Z,E)-9,12-tetradecadienyl acetate, (Z,Z)-9,12-tetradecadienyl acetate, (E,E)-10,12-tetradecadienyl acetate, (E,Z)-10,12-tetradecadienyl acetate, (Z,E)-10,12-tetradecadienyl acetate, (Z,Z)-10,12-tetradecadienyl acetate, (E,Z,Z)-3,8,11-tetradecatrienyl acetate, (E)-8-pentadecenyl acetate, (Z)-8-pentadecenyl acetate, (Z)-9-pentadecenyl acetate, (E)-9-pentadecenyl acetate, (Z)-10-pentadecenyl acetate, (E)-12-pentadecenyl acetate, (Z)-12-pentadecenyl acetate, (Z,Z)-6,9-pentadecadienyl acetate, (E,E)-8,10-pentadecadienyl acetate, (E,Z)-8,10-pentadecadienyl acetate, (Z,E)-8,10-pentadecadienyl acetate, (Z,Z)-8,10-pentadecadienyl acetate, (Z)-3-hexadecenyl acetate, (E)-5-hexadecenyl acetate, (Z)-5-hexadecenyl acetate, (E)-6-hexadecenyl acetate, (E)-7-hexadecenyl acetate, (Z)-7-hexadecenyl acetate, (E)-8-hexadecenyl acetate, (E)-9-hexadecenyl acetate, (Z)-9-hexadecenyl acetate, (Z)-10-hexadecenyl acetate, (E)-11-hexadecenyl acetate, (Z)-11-hexadecenyl acetate, (Z)-12-hexadecenyl acetate, (Z)-14-hexadecenyl acetate, (E,Z)-4,6-hexadecadienyl acetate, (E,Z)-6,11-hexadecadienyl acetate, (Z,Z)-7,10-hexadecadienyl acetate, (Z,E)-7,11-hexadecadienyl acetate, (Z,Z)-7,11-hexadecadienyl acetate, (Z,Z)-8,10-hexadecadienyl acetate, (E,Z)-9,11-hexadecadienyl acetate, (E,E)-10,12-hexadecadienyl acetate, (E,Z)-10,12-hexadecadienyl acetate, (Z,E)-10,12-hexadecadienyl acetate, (E,E)-11,13-hexadecadienyl acetate, (E,Z)-11,13-hexadecadienyl acetate, (Z,E)-11,13-hexadecadienyl acetate, (Z,Z)-11,13-hexadecadienyl acetate, (Z,E)-11,14-hexadecadienyl acetate, (E,E,Z)-4,6,10-hexadecatrienyl acetate, (E,Z,Z)-4,6,10-hexadecatrienyl acetate, (E,E,Z)-4,6,11-hexadecatrienyl acetate, (E,E,E)-10,12,14-hexadecatrienyl acetate, (E,E,Z)-10,12,14-hexadecatrienyl acetate, (E)-8-heptadecenyl acetate, (E)-10-heptadecenyl acetate, (Z)-11-heptadecenyl acetate, (E,E)-4,8-heptadecadienyl acetate, (Z,Z)-8,11-heptadecadienyl acetate, (E)-2-octadecenyl acetate, (Z)-2-octadecenyl acetate, (E)-9-octadecenyl acetate, (Z)-9-octadecenyl acetate, (Z)-11-octadecenyl acetate, (E)-13-octadecenyl acetate, (Z)-13-octadecenyl acetate, (E,Z)-2,13-octadecadienyl acetate, (Z,E)-2,13-octadecadienyl acetate, (Z,Z)-2,13-octadecadienyl acetate, (E,E)-3,13-octadecadienyl acetate, (E,Z)-3,13-octadecadienyl acetate, (Z,E)-3,13-octadecadienyl acetate, (Z,Z)-3,13-octadecadienyl acetate, (E,E)-5,9-octadecadienyl acetate, (Z,Z)-9,12-octadecadienyl acetate, (Z,Z, Z)-3,6,9-octadecatrienyl acetate, and (Z,Z, Z)-9,12,15-octadecatrienyl acetate.
11. The method of claim 1, wherein the recombinant Yarrowia lipolytica microorganism comprises a deletion, disruption, mutation, and/or reduction in the activity of fatty alcohol oxidase YALI0B14014g (FAO1).
12. A method of producing a C6-C24 fatty alcohol, said method comprising: wherein the C6-C24 fatty acyl-CoA is converted to a C6-C24 fatty alcohol by catalytic activity of the fatty alcohol-forming fatty acyl reductase.
- a) providing a recombinant Yarrowia lipolytica microorganism comprising a heterologous nucleic acid molecule encoding and expressing a fatty alcohol-forming fatty acyl reductase comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 75; and
- b) cultivating the recombinant Yarrowia lipolytica of (a) in a culture medium containing a carbon source feedstock and C6-C24 fatty acyl-CoA;
13. The method of claim 12, wherein the fatty alcohol-forming fatty acyl reductase comprises SEQ ID NO: 75.
14. The method of claim 12, wherein the recombinant Yarrowia lipolytica microorganism produces a C6-C24 fatty alcohol selected from the group consisting of (Z)-3-hexen-1-ol, (Z)-3-nonen-1-ol, (E)-2-decen-1-ol, (Z)-2-decen-1-ol, (E)-3-decen-1-ol, (Z)-3-decen-1-ol, (Z)-4-decen-1-ol, (E)-5-decen-1-ol, (Z)-5-decen-1-ol, (E)-8-decen-1-ol, (Z,Z)-4,7-decadien-1-ol, (Z)-3-dodecen-1-ol, (E)-5-dodecen-1-ol, (Z)-5-dodecen-1-ol, (E)-6-dodecen-1-ol, (E)-7-dodecen-1-ol, (Z)-7-dodecen-1-ol, (E)-8-dodecen-1-ol, (Z)-8-dodecen-1-ol, (E)-9-dodecen-1-ol, (Z)-9-dodecen-1-ol, (E)-10-dodecen-1-ol, (Z)-10-dodecen-1-ol, (Z,Z)-3,6-dodecadien-1-ol, (E,E)-5,7-dodecadien-1-ol, (E,Z)-5,7-dodecadien-1-ol, (Z,E)-5,7-dodecadien-1-ol, (E,Z)-7,9-dodecadien-1-ol, (Z,E)-7,9-dodecadien-1-ol, (Z,Z)-7,9-dodecadien-1-ol, (E,E)-8,10-dodecadien-1-ol, (E,Z)-8,10-dodecadien-1-ol, (Z,E)-8,10-dodecadien-1-ol, (Z,Z)-8,10-dodecadien-1-ol, (Z,E,E)-3,6,8-dodecatrien-1-ol, (Z,Z,E)-3,6,8-dodecatrien-1-ol, (Z,Z)-4,7-tridecadien-1-ol, (E)-3-tetradecen-1-ol, (Z)-3-tetradecen-1-ol, (E)-5-tetradecen-1-ol, (Z)-5-tetradecen-1-ol, (E)-7-tetradecen-1-ol, (Z)-7-tetradecen-1-ol, (Z)-8-tetradecen-1-ol, (E)-9-tetradecen-1-ol, (Z)-9-tetradecen-1-ol, (E)-11-tetradecen-1-ol, (Z)-11-tetradecen-1-ol, (Z,Z)-5,8-tetradecadien-1-ol, (E,E)-8,10-tetradecadien-1-ol, (Z,E)-8,10-tetradecadien-1-ol, (Z,E)-9,11-tetradecadien-1-ol, (Z,Z)-9,11-tetradecadien-1-ol, (Z,E)-9,12-tetradecadien-1-ol, (Z,Z)-9,12-tetradecadien-1-ol, (E,E)-10,12-tetradecadien-1-ol, (Z,Z)-10,12-tetradecadien-1-ol, (E)-8-pentadecen-1-ol, (Z)-8-pentadecen-1-ol, (Z,Z)-6,9-pentadecadien-1-ol, (E,Z)-8,10-pentadecadien-1-ol, (E)-5-hexadecen-1-ol, (Z)-5-hexadecen-1-ol, (E)-7-hexadecen-1-ol, (Z)-7-hexadecen-1-ol, (E)-9-hexadecen-1-ol, (Z)-9-hexadecen-1-ol, (E)-10-hexadecen-1-ol, (E)-11-hexadecen-1-ol, (Z)-11-hexadecen-1-ol, (E,E)-1,3-hexadecadien-1-ol, (E,Z)-4,6-hexadecadien-1-ol, (Z,Z)-7,10-hexadecadien-1-ol, (Z,E)-7,11-hexadecadien-1-ol, (Z,Z)-7,11-hexadecadien-1-ol, (E,E)-10,12-hexadecadien-1-ol, (E,Z)-10,12-hexadecadien-1-ol, (E,E)-11,13-hexadecadien-1-ol, (E,Z)-11,13-hexadecadien-1-ol, (Z,E)-11,13-hexadecadien-1-ol, (Z,Z)-11,13-hexadecadien-1-ol, (E,E,Z)-4,6,10-hexadecatrien-1-ol, (E,Z,Z)-4,6,10-hexadecatrien-1-ol, (E)-8-heptadecen-1-ol, (Z)-8-heptadecen-1-ol, (Z)-11-heptadecen-1-ol, (Z,Z)-8,10-heptadecadien-1-ol, (E)-9-octadecen-1-ol, (Z)-9-octadecen-1-ol, (E)-11-octadecen-1-ol, (Z)-11-octadecen-1-ol, (Z)-13-octadecen-1-ol, (E,Z)-2,13-octadecadien-1-ol, (Z,Z)-2,13-octadecadien-1-ol, (E,E)-5,9-octadecadien-1-ol, (E,E)-9,12-octadecadien-1-ol, and (E,E,E)-9,12,15-octadecatrien-1-ol.
15. The method of claim 12, wherein the recombinant Yarrowia lipolytica microorganism comprises a nucleic acid molecule encoding an alcohol oxidase or an alcohol dehydrogenase that catalyzes the conversion of the C6-C24 fatty alcohol into a corresponding C6-C24 fatty aldehyde.
16. The method of claim 15, wherein the alcohol dehydrogenase is selected from the group consisting of Q9NAR7, P14940, Q00669, P21518, P25139, P48584, P22245, Q9NG42, P48585, Q09009, P51549, P21898, Q07588, Q9NG40, P10807, P07162, Q09010, Q00671, Q00672, P07159, P84328, P23361, P23277, Q9U8S9, P23278, Q03384, P28484, P51550, P17648, P48977, P25988, Q00670, P50381, P51552, P42327, P25721, P00334, Q6LCE4, Q24641, P26719, Q50L96, P28483, P51551, Q05114, P37473, Q9GN94, Q0KDL6, P9WQC2, Q8NXU1, P9WQC3, Q7A742, Q2YSX0, Q4J781, Q5HI63, Q2G0G1, Q6GJ63, Q6GBM4, Q2FJ31, Q99W07, Q8CQ56, Q5HRD6, P39462, Q96XE0, P30350, P49645, P48814, Q70UN9, P23991, P86883, P19631, P23236, P48586, P22246, P07161, P12854, Q9Z2M2, Q64413, Q64415, P05336, Q07288, P00333, P86885, P14219, P25141, Q03505, P14673, P80338, P13603, Q07264, P00330, P06525, P00332, Q27404, P00329, P41680, P06757, B4M8Y0, O00097, P41747, Q9P6C8, P43067, Q2R8Z5, P09370, P20369, Q75ZX4, Q5RBP7, P07327, P28469, Q5R1W2, P14139, P00325, P00326, O97959, P48815, Q70UP5, Q70UP6, P27581, P25720, P23237, P48587, P07160, P24267, Q24803, P10847, Q9P4C2, P04707, Q4R1E8, P28032, P14674, O13309, Q96533, P00331, 094038, Q0ITW7, P09369, P49383, 046649, 046650, P10848, P14675, P49384, P07246, P08319, Q9QYY9, Q64563, P80468, P49385, Q09669, A6ZTT5, P10127, Q6XQ67, Q3ZC42, P19854, P28474, P12711, 019053, P11766, Q54TC2, P38113, P28332, P41681, Q5R7Z8, Q5XI95, P40394, P41682, Q64437, P9WQC0, O31186, P9WQC1, P20368, Q7U1B9, P9WQC6, P9WQC7, P0DJA2, F8DVL8, P9WQB8, P9WQB9, P33744, P0A9Q7, P0A9Q8, P72324, A2XAZ3, P39451, P12311, P08843, P54202, P07754, Q9SK86, Q9SK87, A1L4Y2, Q8VZ49, Q0V7W6, Q8LEB2, Q9FH04, P93629, P46415, P71017, Q17335, Q0DWH1, 007737, Q17334, 045687, P33010, P37686, P81786, P80222, P85440, P26325, P80512, P12886, P22797, P25405, P25406, P00327, P00328, P42328, P81600, P81601, P80360, P81431, P80572, P80467, P86884, P79896, A0A0F6Q2K7, A0A0F6Q2W6, AKD01723.1, A0A0F6Q4H2, A0A0F6Q1E8, A0A0F6Q2K3, A0A0F6Q4H7, A0A0F6Q2J9, A0A0F6Q0W0, A0A0F6Q0V0, A0A0F6Q1F1, A0A0F6Q2X2, A0A0F6Q4I2, A0A0F6Q2X0, A0A0F6Q2L9, A0A0F6Q4K1, A0A0F6Q4J7, A0A0F6Q2Y5, A0A0F6Q2Y1, A0A0F6Q1G6, A0A0F6Q2Y9, A0A0F6Q0X5, A0A0F6Q2M3, A0A0F6Q2L1, A0A0F6Q1F9, A0A0F6Q0W6, A0A0F6Q1G9, A0A0F6Q2L4, A0A0F6Q2X6, A0A0F6Q1H3, A0A0F6Q0X1, NP_001188510.1, XP_001655103.1, ETN64198.1, YALI0F09603g (FADH), YALI0D25630g (ADH1), YALI0E17787g (ADH2), YALI0A16379g (ADH3), YALI0E15818g (ADH4), YALI0D02167g (ADH5), YALI0A15147g (ADH6), and YALI0E07766g (ADH7).
17. The method of claim 15, wherein the recombinant Yarrowia lipolytica microorganism produces a C6-C24 fatty aldehyde selected from the group consisting of (E)-2-decenal, (Z)-2-decenal, (Z)-4-decenal, (Z)-5-decenal, (E,E)-2,4-decadienal, (E,Z)-2,4-decadienal, (Z,Z)-2,4-decadienal, (E)-2-undecenal, (E)-2-dodecenal, (Z)-5-dodecenal, (E)-6-dodecenal, (E)-7-dodecenal, (Z)-7-dodecenal, (E)-8-dodecenal, (E)-9-dodecenal, (Z)-9-dodecenal, (E)-10-dodecenal, (E,Z)-5,7-dodecadienal, (Z,E)-5,7-dodecadienal, (Z,Z)-5,7-dodecadienal, (E,Z)-7,9-dodecadienal, (E,E)-8,10-dodecadienal, (E,Z)-8,10-dodecadienal, (Z,E)-8,10-dodecadienal, (Z)-4-tridecenal, (E)-5-tetradecenal, (Z)-5-tetradecenal, (Z)-7-tetradecenal, (Z)-8-tetradecenal, (Z)-9-tetradecenal, (E)-11-tetradecenal, (Z)-11-tetradecenal, (E,E)-2,4-tetradecadienal, (E,Z)-4,9-tetradecadienal, (E,E)-5,8-tetradecadienal, (Z,Z)-5,8-tetradecadienal, (E,E)-8,10-tetradecadienal, (E,Z)-8,10-tetradecadienal, (Z,Z)-8,10-tetradecadienal, (Z,E)-9,11-tetradecadienal, (Z,Z)-9,11-tetradecadienal, (Z,E)-9,12-tetradecadienal, (E,E)-10,12-tetradecadienal, (Z)-10-pentadecenal, (Z,Z)-6,9-pentadecadienal, (E,Z)-9,11-pentadecadienal, (Z,Z)-9,11-pentadecadienal, (E)-7-hexadecenal, (Z)-7-hexadecenal, (E)-9-hexadecenal, (Z)-9-hexadecenal, (E)-10-hexadecenal, (Z)-10-hexadecenal, (E)-11-hexadecenal, (Z)-11-hexadecenal, (Z)-12-hexadecenal, (E)-14-hexadecenal, (E,Z)-4,6-hexadecadienal, (E,Z)-6,11-hexadecadienal, (Z,E)-7,11-hexadecadienal, (Z,Z)-7,11-hexadecadienal, (E,Z)-8,11-hexadecadienal, (E,E)-9,11-hexadecadienal, (E,Z)-9,11-hexadecadienal, (Z,E)-9,11-hexadecadienal, (Z,Z)-9,11-hexadecadienal, (E,E)-10,12-hexadecadienal, (E,Z)-10,12-hexadecadienal, (Z,E)-10,12-hexadecadienal, (Z,Z)-10,12-hexadecadienal, (E,E)-11,13-hexadecadienal, (E,Z)-11,13-hexadecadienal, (Z,E)-11,13-hexadecadienal, (Z,Z)-11,13-hexadecadienal, (E,E)-10,14-hexadecadienal, (E,E,Z)-4,6,11-hexadecatrienal, (Z,Z,E)-7,11,13-hexadecatrienal, (E,E,E)-10,12,14-hexadecatrienal, (E,E,Z)-10,12,14-hexadecatrienal, (E,E,Z,Z)-4,6,11,13-hexadecatetraenal, (E)-2-heptadecenal, (Z)-2-heptadecenal, (Z)-9-heptadecenal, (E)-2-octadecenal, (Z)-2-octadecenal, (E)-9-octadecenal, (Z)-9-octadecenal, (E)-11-octadecenal, (Z)-11-octadecenal, (E)-13-octadecenal, (Z)-13-octadecenal, (E)-14-octadecenal, (E,Z)-2,13-octadecadienal, (E,Z)-3,13-octadecadienal, (Z,Z)-3,13-octadecadienal, (Z,Z)-9,12-octadecadienal, (Z,Z)-11,13-octadecadienal, (E,E)-11,14-octadecadienal, (Z,Z)-13,15-octadecadienal, and (Z,Z, Z)-9,12,15-octadecatrienal.
18. The method of claim 12, wherein the recombinant Yarrowia lipolytica microorganism comprises a nucleic acid molecule encoding an acetyl transferase catalyzes the conversion of the C6-C24 fatty alcohol into a corresponding C6-C24 fatty acetate.
19. The method of claim 18, wherein the acetyl transferase is selected from the group consisting of AY242066, AY242065, AY242064, AY242063, AY242062, EHJ65205, ACX53812, NP_001182381, EHJ65977, EHJ68573, KJ579226, GU594061, KTA99184.1, AIN34693.1, AY605053, XP_002552712.1, XP_503024.1, XP_505595.1, and XP_505513.1.
20. The method of claim 12, wherein the recombinant Yarrowia lipolytica microorganism comprises a nucleic acid molecule encoding an acyl transferase selected from the group consisting of AY242066, AY242065, AY242064, AY242063, AY242062, EHJ65205, ACX53812, NP_001182381, EHJ65977, EHJ68573, KJ579226, GU594061, KTA99184.1, AIN34693.1, AY605053, XP_002552712.1, XP_503024.1, XP_505595.1, XP_505513.1, AAL49962.1, BAC43739.1, AAH89846.1, F6TMU0, F6PXX7, F7B020, ALT83519.1, ANN46862.1, ANN46863.1, ANN46864.1, ANN46865.1, AAC49119.1, JAT48335.1, XP_008793203.1, XP_008806896.1, XP_008806740.1, XP_010908895.1, XP_010908896.1, Q96UY2, A0A077WEU5, A0A068SDP4, A0A068RXA2, A0A197JCE2, A0A1C7N060, I1BLC3, A0A1C7NC56, A0A077X3B5, Q96UY1, A0A077WVD4, A0A163K8G3, S2J8P3, A0A168J818, A0A0C9MR10, A0A162PN39, A0A167QXD0, A0A0C9M4C3, A0A0B7NDT1, A0A015LM78, A0A0B7NHQ3, A0A0A1NVK5, A0A0A1P436, A0A0D7BI48, A0A1B9HZT8, A0A1D1XN50, A0A1B9ILF0, S2JU94, A0A1B9GCB0, A0A068RKT0, Q5KFU4, Q55QC2, U5GY58, A0A197KA94, A0A088FR92, A0A194SBY3, E6R8N8, M7WKS9, A0A191UMW0, C6KZS6, J9VS50, A0A109FM23, I4YE91, A0A066WAJ3, A0A151VHJ4, A0A168LDJ3, A0A0A1ULK8, A0A074RWU7, A0A0K6FWT6, R9AL76, E6ZMU5, A0A0K3CJX4, A0A162Y103, A0A0B7FYU9, A0A1A5ZUI2, A0A1B9GXE9, V5EIP7, A0A127ZHG0, M5FTN9, A0A166HX72, A0A067QH80, A0A165PFB6, G7DXE4, A0A165KJK5, A0A0F7TLQ7, S8FI87, S7ZL04, I2FMX3, F8P370, V2WTH2, S7Q9H4, W3VTZ4, B8M0V7, A0A0D7B6H5, R7SCW4, A0A093UWD0, B6Q8Q9, A0A093VC12, A0A167SF58, A0A180GQ68, E3KWZ5, F4S978, A0A0U5GN87, W9WBT1, A0A0D2A9G0, S3DKQ1, A0A167S691, A0A0C3G1P8, A0A117NM34, A0A0M8NPT1, M2R3J5, A0A1E3JS60, V9DJY4, A0A1C1D128, A0A194XRZ1, A0A135LQY4, F2S034, A0A059J710, R7YTC1, A0A0G4PR11, F2SHG6, A0A022VWY8, A0A178F1Q9, A0A022XM67, F2PHM1, A0A178FDV0, A0A0F8UUV5, A0A0F8XD12, D8Q1Z6, A0A0L0VQ99, W6QE33, A0A0J0XU39, K2RIY7, A0A1B9HIE8, A0A0A2KLE4, A0A177FP94, Q0CU51, A0A0D2C195, K9GS70, K9H4T7, A0A0A2IRX2, A0A165XA55, A0A1E3HS30, R0JHT6, W6XT38, K1WNS8, A0A077R6Q5, A0A0G2F2K4, M2UB23, N4WZB4, A0A0D2ECJ4, K5ULK6, A0A081CNS6, W7E3D1, A0A0D1YAT0, V5FVB4, A0A150V2J4, A0A0D2P224, C5FY83, A0A0E9NND3, A0A163JYI7, M2SYN8, A0A0D2A9Y8, B2WFQ5, A0A178Z686, R1GYF1, A0A0D2AM77, A0A067TPJ7, A0A0G2DT71, A0A0S6XG57, A1CD57, W6ZE59, W9X299, A0A0L1HS74, E3RYE6, A0A178C491, A0A0D2JW30, A0A100ISZ7, G7XRR4, E4ZGH1, A0A0C3AU69, A0A0U1M481, A0A179UDB8, A0A177DML0, A0A074XTA2, R8BK00, A0A178E1M9, A0A074XCF2, A0A178CVL7, J4H349, F2T2H3, T5C9R0, C5GGF5, F8Q4F5, A0A074YHW3, A0A0D2E953, A0A0D2ETM7, A0A163ADJ9, U7PLY5, A0A0F2MF45, A0A0C2J820, A0A1E3B843, A0A0L6WTD3, G2YTS7, W9XGA9, A0A0F4YS69, M9LWR9, A0A074WDM7, M3CBZ0, A0A0C7C2J7, W9YU83, I8IUH8, A0A139HZI0, E9DGY4, A0A0J6F9P8, H6BM52, Q2UDX3, M3ASJ4, A0A177BZU0, A0A017S910, A0A175VVF2, A0A0J8UWI6, A0A0J6YFS7, J3K3F7, A0A0D2FX82, A0A072PSS5, A0A0A1MWE2, W2RSU8, C0S1D5, C1G9R2, A0A1D2JGH6, A0A166PXN0, Q54GC1, A0A0H1B9A9, R4XEF3, D3B2U8, U1HHT8, A0A1E3JYY5, A0A0C3JN41, B6HF05, A0A060S368, K5W449, B0CTA0, F0XD96, and A0A165EP91.
21. The method of claim 18, wherein the recombinant Yarrowia lipolytica microorganism produces a C6-C24 fatty acetate selected from the group consisting of (E)-2-decenyl acetate, (Z)-2-decenyl acetate, (Z)-3-decenyl acetate, (E)-4-decenyl acetate, (Z)-4-decenyl acetate, (E)-5-decenyl acetate, (Z)-5-decenyl acetate, (E)-7-decenyl acetate, (Z)-7-decenyl acetate, (E,E)-3,5-decadienyl acetate, (Z,E)-3,5-decadienyl acetate, (Z,Z)-4,7-decadienyl acetate, (E)-2-undecenyl acetate, (Z)-5-undecenyl acetate, (Z)-7-undecenyl acetate, (Z)-8-undecenyl acetate, (Z)-9-undecenyl acetate, (E)-3-dodecenyl acetate, (Z)-3-dodecenyl acetate, (E)-4-dodecenyl acetate, (E)-5-dodecenyl acetate, (Z)-5-dodecenyl acetate, (Z)-6-dodecenyl acetate, (E)-7-dodecenyl acetate, (Z)-7-dodecenyl acetate, (E)-8-dodecenyl acetate, (Z)-8-dodecenyl acetate, (E)-9-dodecenyl acetate, (Z)-9-dodecenyl acetate, (E)-10-dodecenyl acetate, (Z)-10-dodecenyl acetate, (E,Z)-3,5-dodecadienyl acetate, (Z,E)-3,5-dodecadienyl acetate, (E,E)-4,10-dodecadienyl acetate, (E,E)-5,7-dodecadienyl acetate, (E,Z)-5,7-dodecadienyl acetate, (Z,E)-5,7-dodecadienyl acetate, (Z,Z)-5,7-dodecadienyl acetate, (E,E)-7,9-dodecadienyl acetate, (E,Z)-7,9-dodecadienyl acetate, (Z,E)-7,9-dodecadienyl acetate, (Z,Z)-7,9-dodecadienyl acetate, (E,E)-8,10-dodecadienyl acetate, (E,Z)-8,10-dodecadienyl acetate, (Z,E)-8,10-dodecadienyl acetate, (Z,Z)-8,10-dodecadienyl acetate, (E)-2-tridecenyl acetate, (Z)-2-tridecenyl acetate, (E)-3-tridecenyl acetate, (E)-4-tridecenyl acetate, (Z)-4-tridecenyl acetate, (E)-6-tridecenyl acetate, (Z)-7-tridecenyl acetate, (E)-8-tridecenyl acetate, (Z)-8-tridecenyl acetate, (E)-9-tridecenyl acetate, (Z)-9-tridecenyl acetate, (Z)-10-tridecenyl acetate, (E)-11-tridecenyl acetate, (Z)-11-tridecenyl acetate, (E,Z)-4,7-tridecadienyl acetate, (Z,Z)-4,7-tridecadienyl acetate, (E,Z)-5,9-tridecadienyl acetate, (Z,E)-5,9-tridecadienyl acetate, (Z,Z)-5,9-tridecadienyl acetate, (Z,Z)-7,11-tridecadienyl acetate, (E,Z,Z)-4,7,10-tridecatrienyl acetate, (E)-3-tetradecenyl acetate, (Z)-3-tetradecenyl acetate, (E)-5-tetradecenyl acetate, (Z)-5-tetradecenyl acetate, (E)-6-tetradecenyl acetate, (Z)-6-tetradecenyl acetate, (E)-7-tetradecenyl acetate, (Z)-7-tetradecenyl acetate, (E)-8-tetradecenyl acetate, (Z)-8-tetradecenyl acetate, (E)-9-tetradecenyl acetate, (Z)-9-tetradecenyl acetate, (E)-10-tetradecenyl acetate, (Z)-10-tetradecenyl acetate, (E)-11-tetradecenyl acetate, (Z)-11-tetradecenyl acetate, (E)-12-tetradecenyl acetate, (Z)-12-tetradecenyl acetate, (E,E)-3,5-tetradecadienyl acetate, (E,Z)-3,5-tetradecadienyl acetate, (Z,E)-3,5-tetradecadienyl acetate, (E,Z)-3,7-tetradecadienyl acetate, (E,Z)-3,8-tetradecadienyl acetate, (E,Z)-4,9-tetradecadienyl acetate, (E,Z)-4,10-tetradecadienyl acetate, (Z,Z)-5,8-tetradecadienyl acetate, (E,E)-8,10-tetradecadienyl acetate, (E,Z)-8,10-tetradecadienyl acetate, (Z,E)-8,10-tetradecadienyl acetate, (E,E)-9,11-tetradecadienyl acetate, (E,Z)-9,11-tetradecadienyl acetate, (Z,E)-9,11-tetradecadienyl acetate, (Z,Z)-9,11-tetradecadienyl acetate, (E,E)-9,12-tetradecadienyl acetate, (Z,E)-9,12-tetradecadienyl acetate, (Z,Z)-9,12-tetradecadienyl acetate, (E,E)-10,12-tetradecadienyl acetate, (E,Z)-10,12-tetradecadienyl acetate, (Z,E)-10,12-tetradecadienyl acetate, (Z,Z)-10,12-tetradecadienyl acetate, (E,Z,Z)-3,8,11-tetradecatrienyl acetate, (E)-8-pentadecenyl acetate, (Z)-8-pentadecenyl acetate, (Z)-9-pentadecenyl acetate, (E)-9-pentadecenyl acetate, (Z)-10-pentadecenyl acetate, (E)-12-pentadecenyl acetate, (Z)-12-pentadecenyl acetate, (Z,Z)-6,9-pentadecadienyl acetate, (E,E)-8,10-pentadecadienyl acetate, (E,Z)-8,10-pentadecadienyl acetate, (Z,E)-8,10-pentadecadienyl acetate, (Z,Z)-8,10-pentadecadienyl acetate, (Z)-3-hexadecenyl acetate, (E)-5-hexadecenyl acetate, (Z)-5-hexadecenyl acetate, (E)-6-hexadecenyl acetate, (E)-7-hexadecenyl acetate, (Z)-7-hexadecenyl acetate, (E)-8-hexadecenyl acetate, (E)-9-hexadecenyl acetate, (Z)-9-hexadecenyl acetate, (Z)-10-hexadecenyl acetate, (E)-11-hexadecenyl acetate, (Z)-11-hexadecenyl acetate, (Z)-12-hexadecenyl acetate, (Z)-14-hexadecenyl acetate, (E,Z)-4,6-hexadecadienyl acetate, (E,Z)-6,11-hexadecadienyl acetate, (Z,Z)-7,10-hexadecadienyl acetate, (Z,E)-7,11-hexadecadienyl acetate, (Z,Z)-7,11-hexadecadienyl acetate, (Z,Z)-8,10-hexadecadienyl acetate, (E,Z)-9,11-hexadecadienyl acetate, (E,E)-10,12-hexadecadienyl acetate, (E,Z)-10,12-hexadecadienyl acetate, (Z,E)-10,12-hexadecadienyl acetate, (E,E)-11,13-hexadecadienyl acetate, (E,Z)-11,13-hexadecadienyl acetate, (Z,E)-11,13-hexadecadienyl acetate, (Z,Z)-11,13-hexadecadienyl acetate, (Z,E)-11,14-hexadecadienyl acetate, (E,E,Z)-4,6,10-hexadecatrienyl acetate, (E,Z,Z)-4,6,10-hexadecatrienyl acetate, (E,E,Z)-4,6,11-hexadecatrienyl acetate, (E,E,E)-10,12,14-hexadecatrienyl acetate, (E,E,Z)-10,12,14-hexadecatrienyl acetate, (E)-8-heptadecenyl acetate, (E)-10-heptadecenyl acetate, (Z)-11-heptadecenyl acetate, (E,E)-4,8-heptadecadienyl acetate, (Z,Z)-8,11-heptadecadienyl acetate, (E)-2-octadecenyl acetate, (Z)-2-octadecenyl acetate, (E)-9-octadecenyl acetate, (Z)-9-octadecenyl acetate, (Z)-11-octadecenyl acetate, (E)-13-octadecenyl acetate, (Z)-13-octadecenyl acetate, (E,Z)-2,13-octadecadienyl acetate, (Z,E)-2,13-octadecadienyl acetate, (Z,Z)-2,13-octadecadienyl acetate, (E,E)-3,13-octadecadienyl acetate, (E,Z)-3,13-octadecadienyl acetate, (Z,E)-3,13-octadecadienyl acetate, (Z,Z)-3,13-octadecadienyl acetate, (E,E)-5,9-octadecadienyl acetate, (Z,Z)-9,12-octadecadienyl acetate, (Z,Z, Z)-3,6,9-octadecatrienyl acetate, and (Z,Z, Z)-9,12,15-octadecatrienyl acetate.
22. The method of claim 12, wherein the recombinant Yarrowia lipolytica microorganism comprises a deletion, disruption, mutation, and/or reduction in the activity of fatty alcohol oxidase YALI0B14014g (FAO1).
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Type: Grant
Filed: Aug 20, 2021
Date of Patent: Jan 9, 2024
Patent Publication Number: 20230034938
Assignee: Provivi, Inc. (Santa Monica, CA)
Inventors: Micah Sheppard (Santa Monica, CA), Thomas Heel (Los Angeles, CA), Peter Meinhold (Topanga, CA), Keith Wampler (Santa Monica, CA), Pedro Coelho (Santa Monica, CA), Effendi Leonard (Santa Monica, CA)
Primary Examiner: Tekchand Saidha
Application Number: 17/408,213
